Nucleoside phosphoramidate compound and use thereof

ABSTRACT

A novel nucleoside phosphoramidate compound of the formula below, or a stereoisomer, salt, hydrate, solvate or crystal thereof for the treatment of Flaviviridae family viral infection, especially hepatitis C viral infection, and having a good anti-HCV effect; a pharmaceutical composition having the compound, or a stereoisomer, salt, hydrate, solvate or crystal thereof:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the 35 U.S.C. § 371 National Phase Application ofInternational Application Serial No. PCT/CN2014/073004, filed on Mar. 6,2014, which in turn claims priority to Chinese Patent Application No. CN201310075423.5, filed on Mar. 8, 2013, which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention belongs to the field of pharmaceutical chemistry,and in particular relates to a series of new nucleoside phosphoramidatecompounds, compositions containing the nucleoside phosphoramidatecompounds, and use of said compounds or compositions as the medicamentfor treatment of viral infection diseases, especially as the medicamentfor treatment of viral hepatitis.

BACKGROUND

Hepatitis C virus (HCV) infection is a worldwide epidemic disease, andthere are more than 200 million chronic infected individuals globally.Chronic infection rate of HCV is 15% in Egypt, 4.8% in Pakistan, and3.2% in China, and these countries rank the world top three. Clinicalmanifestations of hepatitis C viral infection are diverse, ranging frommild symptoms such as inflammation to serious illnesses such as livercirrhosis or liver cancer. Chronic hepatitis C can also be complicatedby some extrahepatic manifestations, including rheumatoid arthritis,keratoconjunctivitis sicca, lichen planus, glomerulonephritis, mixedcryoglobulinemia, B-cell lymphoma and porphyria cutanea tarda, etc.,which may be caused by the body's abnormal immune response. Furthermore,various complications can occur during the stage of HCV-inducedcirrhosis decompensation, such as ascites, abdominal infections, uppergastrointestinal bleeding, hepatic encephalopathy, hepatorenal syndrome,liver failure and other symptoms.

HCV belongs to the Flaviviridae family hepatovirus genus, and itsgenetic structure is similar to the other two genera in Flaviviridaefamily, i.e. pestivirus genus and flavivirus genus. Currently, thestandard methods of treatment of HCV infection include interferon alone,and combination therapy of interferon and ribavirin. However, only 50%patients respond to these methods, and interferon has significant sideeffects, such as flu-like symptoms, weight reduction, fatigue andweakness, and combination therapy of interferon and ribavirin hasconsiderable side effects, including hemolysis, anemia and fatigue, etc.

In addition, drugs developed for the treatment of HCV infection includeprotease inhibitors, thiazolidine derivatives, thiazolidines andN-benzanilides, phenan-threnequinone, helicase inhibitors, nucleosidepolymerase inhibitors and gliotoxin, antisense phosphorothioateoligodeoxynucleotides, inhibitors of IRES-dependent translation,ribozymes and nucleoside analogs, etc.

Currently, using nucleoside phosphate compounds for the treatment ofinfection with the virus from the Flaviviridae family, particularly forthe treatment of HCV infection is an important research direction inthis field. WO 2006/065335 disclosed a fluorinatedpyrrolo[2,3,d]pyrimidine nucleoside compound for inhibiting HCV virus.US 2006/0241064 disclosed a nucleoside compound for treating viralinfection caused by the virus from the Flaviviridae family, such as HCV.WO 2008/121634 disclosed a nucleoside phosphoramidate compound fortreating viral infections of mammals.

Notwithstanding the above disclosure, there is still a great need forcompounds effective for the treatment and/or prevention of HCVinfection.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a novel nucleosidephosphoramidate compound of general formula I for the treatment and/orprevention of HCV infection:

or a stereoisomer, salt, hydrate, solvate or crystal thereof,wherein(1) R₁ is selected from C₁₋₆ alkyl;(2) R₂ is selected from halogen;(3) R₃ is selected from OH, H, and C₁₋₄ alkoxy;(4) R₄ is selected from H, C₁₋₆ alkyl, and halogenated C₁₋₆ alkyl;(5) R₅ is selected from C₁₋₆ alkyl, and halogenated C₁₋₆ alkyl;(6) R₆ is selected from the following moieties:

a) phenyl-Y—, wherein Y is absent or selected from C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkenyl-(CO)—, C₂₋₆ alkynyl-(CO)—, O, S,amino and —N(C₁₋₆ alkyl), and wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₆ alkyl, halogen,nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino,halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂and C₁₋₆ alkyl NHCO, or the phenyl and a five- or six-membered ringtaken together form a benzo five-membered ring or benzo six-memberedring;

b) heterocyclyl-Y—, wherein Y is absent or selected from C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkenyl-(CO)—, C₂₋₆ alkynyl-(CO)—, O,S, amino and —N(C₁₋₆ alkyl), or the heterocyclyl together with Y towhich it is attached form a bicyclic heterocycle, and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆ alkyl, halogenated C₁₋₆alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkyl NHCO;

c) C₁₋₆ alkyl-O—C(O)—C₂₋₆ alkenyl- and C₁₋₆ alkyl-O—C(O)—C₂₋₆alkenyl-C(O)—, wherein the C₁₋₆ alkyl is optionally substituted by oneor more groups selected from C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy,cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkylNHCO; and

(7) R₇ is selected from H, halogen, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkoxy, NO₂, CN, C₁₋₆ alkyl-NH—CO—,hydroxy, mono-C₁₋₆ alkylamino, di-C₁₋₆ alkylamino, C₁₋₆ alkyl-S—, C₂₋₆alkenyl-S—, C₂₋₆ alkynyl-S—, C₁₋₆ alkyl-SO—, C₂₋₆ alkenyl-SO—, C₂₋₆alkynyl-SO—, C₁₋₆ alkyl-SO₂—, C₂₋₆ alkenyl-SO₂—, C₂₋₆ alkynyl-SO₂—, C₁₋₆alkyl-OSO₂—, C₂₋₆ alkenyl-OSO₂—, C₂₋₆ alkynyl-OSO₂—; or(8) R₆ and R₇ together with the benzene ring to which they are attachedform a benzo five-membered ring or benzo six-membered ring, wherein thebenzo five-membered ring or benzo six-membered ring is optionallysubstituted by one or more groups selected from halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, phenyl,cyano, C₁₋₆ alkyl-OC(O)— and C₁₋₆ alkyl-OC(O)—CH₂—.

Another objective of the present invention is to provide a preparationmethod of the nucleoside phosphoramidate compound of general formula Ior a stereoisomer, salt, hydrate, solvate or crystal thereof.

Still another objective of the present invention is to provide acomposition comprising a nucleoside phosphoramidate compound of generalformula I of the present invention or a stereoisomer, salt, hydrate,solvate or crystal thereof and a pharmaceutically acceptable carrier,and a composition comprising a nucleoside phosphoramidate compound ofgeneral formula I of the present invention or a stereoisomer, salt,hydrate, solvate or crystal thereof and another antiviral drug.

Still another objective of the present invention is to provide a methodfor treatment and/or prevention of hepatitis C viral infection using anucleoside phosphoramidate compound of general formula I of the presentinvention or a stereoisomer, salt, hydrate, solvate or crystal thereof,as well as use of a nucleoside phosphoramidate compound of generalformula I of the present invention or a stereoisomer, salt, hydrate,solvate or crystal thereof in the manufacture of a medicament fortreatment and/or prevention of hepatitis C viral infection.

For the above objectives, the present invention provides the followingtechnical solutions:

In a first aspect, the present invention provides a novel nucleosidephosphoramidate compound of general formula I:

or a stereoisomer, salt, hydrate, solvate or crystal thereof,wherein(1) R₁ is selected from C₁₋₆ alkyl; preferably, R₁ is selected from C₁₋₃alkyl; more preferably, R₁ is CH₃;(2) R₂ is selected from halogen; preferably, R₂ is F;(3) R₃ is selected from OH, H, C₁₋₄ alkoxy; preferably, R₃ is selectedfrom OH, H, methoxy; more preferably, R₃ is OH;(4) R₄ is selected from H, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl;preferably, R₄ is selected from H, C₁₋₆ alkyl; more preferably, R₄ isselected from H and C₁₋₄ alkyl;(5) R₅ is selected from C₁₋₆ alkyl, halogenated C₁₋₆ alkyl; preferably,R₅ is selected from C₁₋₆ alkyl; more preferably, R₅ is selected fromC₁₋₄ alkyl;(6) R₆ is selected from:

a) phenyl-Y—, wherein Y is absent or selected from C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkenyl-(CO)—, C₂₋₆ alkynyl-(CO)—, O, S,amino and —N(C₁₋₆ alkyl), and wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₆ alkyl, halogen,nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino,halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂and C₁₋₆ alkyl NHCO, or the phenyl and a five- or six-membered ringtaken together form a benzo five-membered ring or benzo six-memberedring;

b) heterocyclyl-Y—, wherein Y is absent or selected from C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkenyl-(CO)—, C₂₋₆ alkynyl-(CO)—, O,S, amino and —N(C₁₋₆ alkyl), or the heterocyclyl together with Y towhich it is attached form a bicyclic heterocycle, and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆ alkyl, halogenated C₁₋₆alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkyl NHCO;

c) C₁₋₆ alkyl-O—C(O)—C₂₋₆ alkenyl- and C₁₋₆ alkyl-O—C(O)—C₂₋₆alkenyl-C(O)—, wherein the C₁₋₆ alkyl is optionally substituted by oneor more groups selected from C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy,cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkylNHCO;

preferably, R₆ is selected from:

a) phenyl-Y—, wherein Y is absent or selected from C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl-(CO)—, C₂₋₄ alkynyl-(CO)—, O, S,amino and —N(C₁₋₄ alkyl), and wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₄ alkyl, halogen,nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino,halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂and C₁₋₄ alkyl NHCO, or the phenyl and a five- or six-membered ringtaken together form a benzo five-membered ring or benzo six-memberedring;

b) heterocyclyl-Y—, wherein Y is absent or selected from C₁₋₄ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl-(CO)—, C₂₋₄ alkynyl-(CO)—, O,S, amino and —N(C₁₋₄ alkyl), or the heterocyclyl together with Y towhich it is attached form a bicyclic heterocycle, and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO;

c) C₁₋₄ alkyl-O—C(O)—C₂₋₄ alkenyl- and C₁₋₄ alkyl-O—C(O)—C₂₋₄alkenyl-C(O)—, wherein the C₁₋₄ alkyl is optionally substituted by oneor more groups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy,cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkylNHCO;

more preferably, R₆ is selected from:

a) phenyl, phenyl-C₁₋₃ alkyl-, phenyl-C₂₋₃ alkenyl-, phenyl-C₂₋₃alkynyl-, phenyl-O—, phenyl-S—, phenyl-NH—, phenyl-N(C₁₋₃ alkyl)-,phenyl-ethenyl-(CO)— and naphthyl-ethenyl-(CO)—, wherein the phenyl isoptionally substituted by one or more groups selected from C₁₋₄ alkyl,halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino,N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO, or the phenyl and a group selectedfrom phenyl, oxazolyl, pyrazinyl and pyrrolyl taken together form anaphthyl, benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl;

b) 1H-imidazolyl-Y—, 1,2,4-triazolyl-Y—, 1,2,3-triazolyl-Y—,thiazolyl-Y—, 1,2,3-thiadiazolyl-Y—, 1,2,4-thiadiazolyl-Y—,1,3,4-thiadiazolyl-Y—, oxazolyl-Y—, 1,2,4-oxadiazolyl-Y—,1,2,3-oxadiazolyl-Y—, 1,3,4-oxadiazolyl-Y—, pyrimidinyl-Y—,pyrazinyl-Y—, pyridazinyl-Y—, quinoxalinyl-Y—, 4H-chromen-4-one-Y—,pyridyl-Y—, thienyl-Y—, thieno[3,2-b]thienyl-Y—, wherein Y is absent orselected from methyl, ethyl, ethenyl, ethynyl, ethenyl-(CO)—,ethynyl-(CO)—, O, S, amino and —NCH₂—, and wherein each of theheterocyclyl groups is optionally substituted by one or more groupsselected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO;

c) methyl-OC(O)—═—, ethyl-OC(O)—═—, propyl-OC(O)—═—, isopropyl-OC(O)—═—,butyl-OC(O)—═—, isobutyl-OC(O)—═— and t-butyl-OC(O)—═—.

Still more preferably, R₆ is selected from:

a) phenyl, benzoxazolyl, benzo[b]pyrazinyl, benzo[b]pyrrolyl,phenyl-(CH₂)—, phenyl-=-, phenyl-=-C(O)— and phenyl-≡-, wherein thephenyl is optionally substituted by one or more groups selected fromC₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO;

b) 1H-imidazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, thiazolyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, oxazolyl,1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl, pyrimidinyl,pyrazinyl, pyridazinyl, quinoxalinyl, 4H-chromen-4-one, pyridyl,thieno[3,2-b]thienyl, 1H-imidazolyl-(CH₂)—, 1,2,4-triazolyl-(CH₂)—,1,2,3-triazolyl-(CH₂)—, thiazolyl-(CH₂)—, 1,2,3-thiadiazolyl-(CH₂)—,1,2,4-thiadiazolyl-(CH₂)—, 1,3,4-thiadiazolyl-(CH₂)—, oxazolyl-(CH₂)—,1,2,4-oxadiazolyl-(CH₂)—, 1,2,3-oxadiazolyl-(CH₂)—,1,3,4-oxadiazolyl-(CH₂)—, pyrimidinyl-(CH₂)—, pyrazinyl-(CH₂)—,pyridazinyl-(CH₂)—, quinoxalinyl-(CH₂)—, 4H-chromen-4-one-(CH₂)—,pyridyl-(CH₂)—, thieno[3,2-b]thienyl-(CH₂)—, 1H-imidazolyl-=-,1,2,4-triazolyl-=-, 1,2,3-triazolyl-=-, thiazolyl-=-,1,2,3-thiadiazolyl-=-, 1,2,4-thiadiazolyl-=-, 1,3,4-thiadiazolyl-=-,oxazolyl-=-, 1,2,4-oxadiazolyl-=-, 1,2,3-oxadiazolyl-=-,1,3,4-oxadiazolyl-=-, pyrimidinyl-=-, pyrazinyl-=-, pyridazinyl-=-,quinoxalinyl-=-, 4H-chromen-4-one-=-, pyridyl-=-,thieno[3,2-b]thienyl-=-, 1H-imidazolyl-≡-, 1,2,4-triazolyl-≡-,1,2,3-triazolyl-≡-, thiazolyl-≡-, 1,2,3-thiadiazolyl-≡-,1,2,4-thiadiazolyl-≡-, 1,3,4-thiadiazolyl-≡-, oxazolyl-≡-,1,2,4-oxadiazolyl-≡-, 1,2,3-oxadiazolyl-≡-, 1,3,4-oxadiazolyl-≡-,pyrimidinyl-≡-, pyrazinyl-≡-, pyridazinyl-≡-, quinoxalinyl-≡-,4H-chromen-4-one-≡-, pyridyl-≡- and thieno[3,2-b]thienyl-≡-, whereineach of heterocyclyl groups is optionally substituted by one or moregroups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO;

c) methyl-OC(O)—═—; and

(7) R₇ is selected from H, halogen, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkoxy, NO₂, CN, C₁₋₆ alkyl-NH—CO—,hydroxy, mono-C₁₋₆ alkylamino, di-C₁₋₆ alkylamino, C₁₋₆ alkyl-S—, C₂₋₆alkenyl-S—, C₂₋₆ alkynyl-S—, C₁₋₆ alkyl-SO—, C₂₋₆ alkenyl-SO—, C₂₋₆alkynyl-SO—, C₁₋₆ alkyl-SO₂—, C₂₋₆ alkenyl-SO₂—, C₂₋₆ alkynyl-SO₂—, C₁₋₆alkyl-OSO₂—, C₂₋₆ alkenyl-OSO₂—, C₂₋₆ alkynyl-OSO₂—; preferably, R₇ isselected from H, halogen, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl, C₁₋₆alkoxy, halogenated C₁₋₆ alkoxy, NO₂, CN, C₁₋₆ alkyl-NH—CO—; morepreferably, R₇ is selected from H, halogen, C₁₋₄ alkyl, halogenated C₁₋₄alkyl, C₁₋₄ alkoxy, halogenated C₁₋₄ alkoxy, NO₂, CN, C₁₋₄ alkyl-NH—CO—;or(8) R₆ and R₇ together with the benzene ring to which they are attachedform a benzo five-membered ring or benzo six-membered ring, wherein thebenzo five-membered ring or benzo six-membered ring is optionallysubstituted by one or more groups selected from halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, phenyl,cyano, C₁₋₆ alkyl-OC(O)—, C₁₋₆ alkyl-OC(O)—CH₂—; preferably, R₆ and R₇together with the benzene ring to which they are attached form a1,2,3,4-tetrahydronaphthalene, 2,3-dihydro-1H-indene, indole,benzofuran, quinoxaline, 4H-chromen-4-one, benzo[d]isoxazole,benzo[d]oxazole, benzo[c][1,2,5]thiadiazole, benzo[b]thiophene,benzodihydropyran-4-one, wherein each of these fused rings is optionallysubstituted by one or more groups selected from halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, phenyl,cyano, C₁₋₆ alkyl-OC(O)— and C₁₋₆ alkyl-OC(O)—CH₂—.

In a preferred embodiment, the present invention provides a compound ofgeneral formula I or a stereoisomer, salt, hydrate, solvate or crystalthereof, wherein R₁ is methyl, ethyl, propyl or isopropyl; R₂ is F; R₃is OH, H or methoxy; R₄ is H, methyl, ethyl, n-propyl or isopropyl; R₅is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl; R₆is phenyl-Y— or heterocyclyl-Y—, wherein Y is absent or selected fromC₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)—, C₂₋₃alkynyl-(CO)—, O, S, amino and —N(C₁₋₃ alkyl), and wherein the phenyl orthe heterocyclyl is optionally substituted by one or more groupsselected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃ alkoxy, cyano, C₂₋₃alkenyl, C₂₋₃ alkynyl, C₁₋₃ acylamino, halogenated C₁₋₃ alkyl,halogenated C₁₋₃ alkoxy, amino, N(C₁₋₃ alkyl)₂ and C₁₋₃ alkyl NHCO, orthe phenyl and a five- or six-membered ring taken together form a benzofive-membered ring or benzo six-membered ring.

Surprisingly, the inventors of the present invention have found that,when R₆ group and the oxygen group both of which are attached to thebenzene ring in general formula I are at the para- or meta-position toeach other, especially where R₆ is selected from phenyl-Y— andheterocyclyl-Y—, the compound according to the present invention has avery excellent anti-HCV activity. Without wishing to be bound by anyexisting theory, the inventors of the present invention believe that,the compound of general formula I wherein R₆ group particularly selectedfrom phenyl-Y—, and the phenyl-Y— or heterocyclyl-Y— is positionedeither para or meta to the —O— group attached to the benzene ring in thestructure of phenyl-Y-phenyl-O— or heterocyclyl-Y-phenyl-O—, exhibitsexcellent anti-HCV activity.

In a further preferred embodiment, the present invention provides acompound of general formula I or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₁ is methyl or ethyl; R₂ is F; R₃ is OH orH; R₄ is H, methyl or ethyl; R₅ is methyl, ethyl, n-propyl or isopropyl;R₆ is phenyl-Y—, wherein Y is absent or selected from C₁₋₃ alkyl, C₂₋₃alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)— and C₂₋₃ alkynyl-(CO)—, andwherein the phenyl is optionally substituted by one or more groupsselected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃ alkoxy, cyano, C₂₋₃alkenyl, C₂₋₃ alkynyl, C₁₋₃ acylamino, halogenated C₁₋₃ alkyl,halogenated C₁₋₃ alkoxy, amino, N(C₁₋₃ alkyl)₂ and C₁₋₃ alkyl NHCO, orthe phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyltaken together form a benzoxazolyl, benzo[b]pyrazinyl orbenzo[b]pyrrolyl, wherein R₆ group “phenyl-Y—” and the oxygen group bothof which are attached to the benzene ring are at the para- ormeta-position to each other.

In a further preferred embodiment, the present invention provides acompound of general formula I or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₁ is methyl; R₂ is F; R₃ is OH; R₄ is H,methyl or ethyl; R₅ is methyl, ethyl, n-propyl or isopropyl; R₆ isphenyl-Y—, wherein Y is absent or selected from C₁₋₃ alkyl, C₂₋₃alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)— and C₂₋₃ alkynyl-(CO)—, andwherein the phenyl is optionally substituted by one or more groupsselected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃ alkoxy, halogenated C₁₋₃alkyl, halogenated C₁₋₃ alkoxy, cyano, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₁₋₃acylamino, amino and C₁₋₃ alkyl NHCO, or the phenyl and a group selectedfrom oxazolyl, pyrazinyl and pyrrolyl taken together form abenzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R₆ group“phenyl-Y—” and the oxygen group both of which are attached to thebenzene ring are at the para- or meta-position to each other.

In a further preferred embodiment, the present invention provides acompound of general formula I or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₁ is methyl; R₂ is F; R₃ is OH; R₄ is H ormethyl; R₅ is isopropyl; R₆ is phenyl-Y—, wherein Y is absent orselected from C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)—and C₂₋₃ alkynyl-(CO)—, and wherein the phenyl is optionally substitutedby one or more groups selected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃alkoxy, halogenated C₁₋₃ alkyl, halogenated C₁₋₃ alkoxy, cyano and C₁₋₃alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyland pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl orbenzo[b]pyrrolyl, wherein R₆ group “phenyl-Y—” and the oxygen group bothof which are attached to the benzene ring are at the para- ormeta-position to each other.

In a further preferred embodiment, the present invention provides acompound of general formula I or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₆ is phenyl or benzyl, preferably, thephenyl or benzyl and the oxygen group both of which are attached to thebenzene ring are at the para- or meta-position to each other.

In other embodiments, the present invention provides a compound ofgeneral formula I or a stereoisomer, salt, hydrate, solvate or crystalthereof, wherein R₆ is heterocyclyl-Y—, wherein the heterocyclyl canalso be furyl, pyrrolyl, tetrazolyl, furazanyl, dioxadiazolyl, pyranyl,thiopyranyl, piperidinyl, triazinyl, oxazinyl, etc.

In some embodiments, more preferably, the present invention provides acompound of general formula Ia:

or a stereoisomer, salt, hydrate, solvate or crystal thereof,wherein(1) R₆ is selected from:

a) phenyl-Y—, wherein Y is absent or selected from C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkynyl-(CO)—, C₂₋₆ alkynyl-(CO)—, O, S,amino and —N(C₁₋₆ alkyl), and wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₆ alkyl, halogen,nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino,halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂and C₁₋₆ alkyl NHCO, or the phenyl and a five- or six-membered ringtaken together form a benzo five-membered ring or benzo six-memberedring;

b) heterocyclyl-Y—, wherein Y is absent or selected from C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkenyl-(CO)—, C₂₋₆ alkynyl-(CO)—, O,S, amino and —N(C₁₋₆ alkyl), or the heterocyclyl together with Y towhich it is attached form a bicyclic heterocycle, and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆ alkyl, halogenated C₁₋₆alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkyl NHCO; and

c) C₁₋₆ alkyl-O—C(O)—C₂₋₆ alkenyl- and C₁₋₆ alkyl-O—C(O)—C₂₋₆alkenyl-C(O)—, wherein the C₁₋₆ alkyl is optionally substituted by oneor more groups selected from C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy,cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkylNHCO;

preferably, R₆ is selected from:

a) phenyl-Y—, wherein Y is absent or selected from C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl-(CO)—, C₂₋₄ alkynyl-(CO)—, O, S,amino and —N(C₁₋₄ alkyl), and wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₄ alkyl, halogen,nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino,halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂and C₁₋₄ alkyl NHCO, or the phenyl and a five- or six-membered ringtaken together form a benzo five-membered ring or benzo six-memberedring;

b) heterocyclyl-Y—, wherein Y is absent or selected from C₁₋₄ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₂₋₄ alkenyl-(CO)—, C₂₋₄ alkynyl-(CO)—, O,S, amino and —N(C₁₋₄ alkyl), or the heterocyclyl together with Y towhich it is attached form a bicyclic heterocycle, and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; and

c) C₁₋₄ alkyl-O—C(O)—C₂₋₄ alkenyl- and C₁₋₄ alkyl-O—C(O)—C₂₋₄alkenyl-C(O)—, wherein the C₁₋₄ alkyl is optionally substituted by oneor more groups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy,cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkylNHCO;

more preferably, R₆ is selected from:

a) phenyl, phenyl-C₁₋₃ alkyl-, phenyl-C₂₋₃ alkenyl-, phenyl-C₂₋₃alkynyl-, phenyl-O—, phenyl-S—, phenyl-NH—, phenyl-N(C₁₋₃ alkyl)-,phenyl-ethenyl-(CO)— and naphthyl-ethenyl-(CO)—, wherein the phenyl isoptionally substituted by one or more groups selected from C₁₋₄ alkyl,halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino,N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO, or the phenyl and a group selectedfrom phenyl, oxazolyl, pyrazinyl and pyrrolyl taken together form anaphthyl, benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl;

b) 1H-imidazolyl-Y—, 1,2,4-triazolyl-Y—, 1,2,3-triazolyl-Y—,thiazolyl-Y—, 1,2,3-thiadiazolyl-Y—, 1,2,4-thiadiazolyl-Y—,1,3,4-thiadiazolyl-Y—, oxazolyl-Y—, 1,2,4-oxadiazolyl-Y—,1,2,3-oxadiazolyl-Y—, 1,3,4-oxadiazolyl-Y—, pyrimidinyl-Y—,pyrazinyl-Y—, pyridazinyl-Y—, quinoxalinyl-Y—, 4H-chromen-4-one-Y—,pyridyl-Y—, thienyl-Y—, thieno[3,2-b]thienyl-Y—, wherein Y is absent orselected from —CH₂—, —CH₂—CH₂—, ethenyl, ethynyl, ethenyl-(CO)—,ethynyl-(CO)—, O, S, amino and —NCH₂—, wherein each of the heterocyclylgroups is optionally substituted by one or more groups selected fromC₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; and

c) methyl-OC(O)—═—, ethyl-OC(O)—═—, propyl-OC(O)—═—, isopropyl-OC(O)—═—,butyl-OC(O)—═—, isobutyl-OC(O)—═— and t-butyl-OC(O)—═—;

Still more preferably, R₆ is selected from:

a) phenyl, phenyl-(CH₂)—, phenyl-=-, phenyl-=-C(O)— and phenyl-≡-,wherein the phenyl is optionally substituted by one or more groupsselected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, amino,N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO, or the phenyl and a group selectedfrom oxazolyl, pyrazinyl and pyrrolyl taken together form abenzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl;

b) 1H-imidazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, thiazolyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, oxazolyl,1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl, pyrimidinyl,pyrazinyl, pyridazinyl, quinoxalinyl, 4H-chromen-4-one,thieno[3,2-b]thienyl, 1H-imidazolyl-(CH₂)—, 1,2,4-triazolyl-(CH₂)—,1,2,3-triazolyl-(CH₂)—, thiazolyl-(CH₂)—, 1,2,3-thiadiazolyl-(CH₂)—,1,2,4-thiadiazolyl-(CH₂)—, 1,3,4-thiadiazolyl-(CH₂)—, oxazolyl-(CH₂)—,1,2,4-oxadiazolyl-(CH₂)—, 1,2,3-oxadiazolyl-(CH₂)—,1,3,4-oxadiazolyl-(CH₂)—, pyrimidinyl-(CH₂)—, pyrazinyl-(CH₂)—,pyridazinyl-(CH₂)—, quinoxalinyl-(CH₂)—, 4H-chromen-4-one-(CH₂)—,pyridyl-(CH₂)—, thieno[3,2-b]thienyl-(CH₂)—, 1H-imidazolyl-=-,1,2,4-triazolyl-=-, 1,2,3-triazolyl-=-, thiazolyl-=-,1,2,3-thiadiazolyl-=-, 1,2,4-thiadiazolyl-=-, 1,3,4-thiadiazolyl-=-,oxazolyl-=-, 1,2,4-oxadiazolyl-=-, 1,2,3-oxadiazolyl-=-,1,3,4-oxadiazolyl-=-, pyrimidinyl-=-, pyrazinyl-=-, pyridazinyl-=-,quinoxalinyl-=-, 4H-chromen-4-one-=-, pyridyl-=-,thieno[3,2-b]thienyl-=-, 1H-imidazolyl-≡-, 1,2,4-triazolyl-≡-,1,2,3-triazolyl-≡-, thiazolyl-≡-, 1,2,3-thiadiazolyl-≡-,1,2,4-thiadiazolyl-≡-, 1,3,4-thiadiazolyl-≡-, oxazolyl-≡-,1,2,4-oxadiazolyl-≡-, 1,2,3-oxadiazolyl-≡-, 1,3,4-oxadiazolyl-≡-,pyrimidinyl-≡-, pyrazinyl-≡-, pyridazinyl-≡-, quinoxalinyl-≡-,4H-chromen-4-one-≡-, pyridyl-≡- and thieno[3,2-b]thienyl-≡-, whereineach of the heterocyclyl groups is optionally substituted by one or moregroups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; and

c) methyl-OC(O)—═—; and

(2) R₇ is selected from H, halogen, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkoxy, NO₂, CN and C₁₋₆ alkyl-NH—CO—;preferably, R₇ is selected from H, halogen, C₁₋₄ alkyl, halogenated C₁₋₄alkyl, C₁₋₄ alkoxy, halogenated C₁₋₄ alkoxy, NO₂, CN and C₁₋₄alkyl-NH—CO—; more preferably, R₇ is selected from H, fluoro, chloro,methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl,trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, methoxy,ethoxy, n-propoxy, isopropoxy, fluoromethoxy, difluoromethoxy,trifluoromethoxy, fluoroethoxy, difluoroethoxy, trifluoroethoxy, NO₂, CNand C₁₋₄ alkyl-NH—CO—; or(3) R₆ and R₇ together with the benzene ring to which they are attachedform a benzo five-membered ring or benzo six-membered ring, wherein thebenzo five-membered ring or benzo six-membered ring is optionallysubstituted by one or more groups selected from halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, phenyl,cyano, C₁₋₆ alkyl-OC(O)—, C₁₋₆ alkyl-OC(O)—CH₂—; preferably, R₆ and R₇together with the benzene ring to which they are attached form a1,2,3,4-tetrahydronaphthalene, 2,3-dihydro-1H-indene, indole,benzofuran, quinoxaline, 4H-chromen-4-one, benzo[d]isoxazole,benzo[d]oxazole, benzo[c][1,2,5]thiadiazole, benzo[b]thiophene,benzodihydropyran-4-one, wherein each of these fused rings is optionallysubstituted by one or more groups selected from halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, halogenated C₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, phenyl,cyano, C₁₋₆ alkyl-OC(O)—, C₁₋₆ alkyl-OC(O)—CH₂—.

In a preferred embodiment, the present invention provides a compound ofgeneral formula Ia or a stereoisomer, salt, hydrate, solvate or crystalthereof, wherein R₆ is phenyl-Y— or heterocyclyl-Y—, wherein Y is absentor selected from C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₂₋₃alkenyl-(CO)—, C₂₋₃ alkynyl-(CO)—, O, S, amino and —N(C₁₋₃ alkyl), andwherein the phenyl or heterocyclyl is optionally substituted by one ormore groups selected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃ alkoxy,cyano, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₁₋₃ acylamino, halogenated C₁₋₃alkyl, halogenated C₁₋₃ alkoxy, amino, NH(C₁₋₃ alkyl), N(C₁₋₃ alkyl)₂and C₁₋₃ alkyl NHCO, or the phenyl and a five- or six-membered ringtaken together form a benzo five-membered ring or benzo six-memberedring. Preferably, the present invention provides the compound of generalformula Ia or a stereoisomer, salt, hydrate, solvate or crystal thereof,wherein R₆ group “phenyl-Y—” or “heterocyclyl-Y—” and the oxygen groupboth of which are attached to the benzene ring are at the para- ormeta-position to each other.

The present invention provides the compound of general formula Ia or astereoisomer, salt, hydrate, solvate or crystal thereof, wherein R₆ is1H-imidazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, thiazolyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, oxazolyl,1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl, pyrimidinyl,pyrazinyl, pyridazinyl, quinoxalinyl, 4H-chromen-4-one,thieno[3,2-b]thienyl, 1H-imidazolyl-(CH₂)—, 1,2,4-triazolyl-(CH₂)—,1,2,3-triazolyl-(CH₂)—, thiazolyl-(CH₂)—, 1,2,3-thiadiazolyl-(CH₂)—,1,2,4-thiadiazolyl-(CH₂)—, 1,3,4-thiadiazolyl-(CH₂)—, oxazolyl-(CH₂)—,1,2,4-oxadiazolyl-(CH₂)—, 1,2,3-oxadiazolyl-(CH₂)—,1,3,4-oxadiazolyl-(CH₂)—, pyrimidinyl-(CH₂)—, pyrazinyl-(CH₂)—,pyridazinyl-(CH₂)—, quinoxalinyl-(CH₂)—, 4H-chromen-4-one-(CH₂)—,pyridyl-(CH₂)—, thieno[3,2-b]thienyl-(CH₂)—, 1H-imidazolyl-=-,1,2,4-triazolyl-=-, 1,2,3-triazolyl-=-, thiazolyl-=-,1,2,3-thiadiazolyl-=-, 1,2,4-thiadiazolyl-=-, 1,3,4-thiadiazolyl-=-,oxazolyl-=-, 1,2,4-oxadiazolyl-=-, 1,2,3-oxadiazolyl-=-,1,3,4-oxadiazolyl-=-, pyrimidinyl-=-, pyrazinyl-=-, pyridazinyl-=-,quinoxalinyl-=-, 4H-chromen-4-one-=-, pyridyl-=-,thieno[3,2-b]thienyl-=-, 1H-imidazolyl-≡-, 1,2,4-triazolyl-≡-,1,2,3-triazolyl-≡-, thiazolyl-≡-, 1,2,3-thiadiazolyl-≡-,1,2,4-thiadiazolyl-≡-, 1,3,4-thiadiazolyl-≡-, oxazolyl-≡-,1,2,4-oxadiazolyl-≡-, 1,2,3-oxadiazolyl-≡-, 1,3,4-oxadiazolyl-≡-,pyrimidinyl-≡-, pyrazinyl-≡-, pyridazinyl-≡-, quinoxalinyl-≡-,4H-chromen-4-one-≡-, pyridyl-≡-, or thieno[3,2-b]thienyl-≡-, whereineach of these heterocyclyl groups is optionally substituted by one ormore groups selected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃ alkoxy,cyano, halogenated C₁₋₃ alkyl, halogenated C₁₋₃ alkoxy, C₂₋₃ alkenyl,C₂₋₃ alkynyl, C₁₋₃ acylamino, amino, NH(C₁₋₃ alkyl), N(C₁₋₃ alkyl)₂ andC₁₋₄ alkyl NHCO, wherein the heterocyclyl and the oxygen group both ofwhich are attached to the benzene ring are at the para- or meta-positionto each other.

In a further preferred embodiment, the present invention provides acompound of general formula Ia or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₆ is phenyl-Y—, wherein Y is absent orselected from C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)—and C₂₋₃ alkynyl-(CO)—, and wherein the phenyl is optionally substitutedby one or more groups selected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃alkoxy, cyano, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₁₋₃ acylamino, halogenatedC₁₋₃ alkyl, halogenated C₁₋₃ alkoxy, amino, NH(C₁₋₃ alkyl), N(C₁₋₃alkyl)₂ and C₁₋₃ alkyl NHCO, or the phenyl and a group selected fromoxazolyl, pyrazinyl and pyrrolyl taken together form a benzoxazolyl,benzo[b]pyrazinyl or benzo[b]pyrrolyl, wherein R₆ group and the oxygengroup both of which are attached to the benzene ring are at the para- ormeta-position to each other.

In a further preferred embodiment, the present invention provides acompound of general formula Ia or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₆ is phenyl-Y—, wherein Y is absent orselected from C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)—and C₂₋₃ alkynyl-(CO)—, and wherein the phenyl is optionally substitutedby one or more groups selected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃alkoxy, cyano, C₂₋₃ alkenyl, C₂₋₃ alkynyl, halogenated C₁₋₃ alkyl,halogenated C₁₋₃ alkoxy, C₁₋₃ acylamino, amino and C₁₋₃ alkyl NHCO, orthe phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyltaken together form a benzoxazolyl, benzo[b]pyrazinyl orbenzo[b]pyrrolyl, wherein R₆ group and the oxygen group both of whichare attached to the benzene ring are at the para- or meta-position toeach other.

In a further preferred embodiment, the present invention provides acompound of general formula Ia or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₆ is phenyl-Y—, wherein Y is absent orselected from C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₂₋₃ alkenyl-(CO)—and C₂₋₃ alkynyl-(CO)—, and wherein the phenyl is optionally substitutedby one or more groups selected from C₁₋₃ alkyl, halogen, nitro, C₁₋₃alkoxy, cyano, halogenated C₁₋₃ alkyl, halogenated C₁₋₃ alkoxy and C₁₋₃alkyl NHCO, or the phenyl and a group selected from oxazolyl, pyrazinyland pyrrolyl taken together form a benzoxazolyl, benzo[b]pyrazinyl orbenzo[b]pyrrolyl, wherein R₆ group and the oxygen group both of whichare attached to the benzene ring are at the para- or meta-position toeach other.

In a further preferred embodiment, the present invention provides acompound of general formula Ia or a stereoisomer, salt, hydrate, solvateor crystal thereof, wherein R₆ is phenyl- or benzyl- (i.e.,phenyl-CH₂—), preferably, the phenyl or benzyl and the oxygen group bothof which are attached to the benzene ring are at the para- ormeta-position to each other.

In other embodiments, the present invention provides a compound ofgeneral formula Ia or a stereoisomer, salt, hydrate, solvate or crystalthereof, wherein R₆ is heterocyclyl-Y—, wherein the heterocyclyl canalso be furyl, pyrrolyl, tetrazolyl, furazanyl, dioxadiazolyl, pyranyl,thiopyranyl, piperidinyl, triazinyl, oxazinyl, etc.

The present invention provides the following specific compounds:

In another aspect, the present invention provides a preparation methodof a compound of general formula I or Ia according to the presentinvention, comprising the following steps:

a) reacting compound (1) with phosphorus oxychloride under an alkinecondition, followed by adding compound (2), and then addingpentafluorophenol, to obtain compound (3);b) reacting compound (3) with compound (4) at low temperature, to givethe subject compound (5).wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are as defined in general formulaI above.

In a third aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of general formula I or Ia accordingto the present invention, or a stereoisomer, salt, hydrate, solvate orcrystal thereof.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a compound of general formula I or Ia, or astereoisomer, salt, hydrate, solvate or crystal thereof, and additionalanti-HCV therapeutic agents selected from one or more of the followings:HCV NS3 protease inhibitors, HCV NS5B RNA-dependent RNA polymeraseinhibitors, nucleoside analogs, interferon α, pegylated interferon,ribavirin, levovirin, viramidine, TLR7 agonists, TLR9 agonists,cyclophilin inhibitors, α-glucosidase inhibitors, NS5A inhibitors andNS3 helicase inhibitors.

A pharmaceutical formulation may be formulated by mixing the compound offormula I or Ia of the present invention, or a stereoisomer, salt,hydrate, solvate or crystal thereof with a pharmaceutically acceptablecarrier, diluent or excipient for suitable for oral or parenteraladministration. Methods of administration include, but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal and oral routes. The formulations may beadministered by any routes, for example by infusion or bolus injection,or via epithelial or mucocutaneous (such as oral or rectal mucosa, etc.)absorption. The administration may be systematic or topical. Examples offormulations for oral administration include solid or liquid dosageforms, specifically, comprising tablets, pills, granules, powder,capsules, syrups, emulsions, suspensions and the like. The formulationsmay be prepared by methods well known in the art, and may compriseconventional carriers, diluents or excipients in the field ofpharmaceutical formulation.

In a fourth aspect, the present invention provides a method of treatinga subject infected with virus from the Flaviviridae family using acompound of general formula I or Ia of the present invention, or astereoisomer, salt, hydrate, solvate or crystal thereof or apharmaceutical composition of the present invention, comprisingadministering a compound of general formula I or Ia, or a stereoisomer,salt, hydrate, solvate or crystal thereof or a pharmaceuticalcomposition comprising a compound of general formula I or Ia, or astereoisomer, salt, hydrate, solvate or crystal thereof to the subjectin an amount of effectively reducing the viral load of said virus insaid subject. In one embodiment, the present invention provides a methodof treating and/or preventing RNA viral infection, e.g. Flaviviridaefamily viral infection, comprising administering a compound of thepresent invention, or a stereoisomer, salt, hydrate, solvate, crystal ora pharmaceutical composition thereof to a subject in need thereof. Inanother embodiment, the present invention provides a method ofinhibiting RNA viral infection, e.g. Flaviviridae family viralinfection, comprising contacting said virus with a therapeuticallyeffective amount of a compound of the present invention, or astereoisomer, salt, hydrate, solvate, crystal or a pharmaceuticalcomposition thereof.

“Flaviviridae viruses” means any virus of the Flaviviridae family,including those viruses that infect human or non-human animals, such asflavivirus, pestivirus and hepatitis C virus. The compounds andcompositions of the present invention may be used particularly for thetreatment or preventive treatment of HCV infection.

In another aspect, the present invention provides a use of a compound ofgeneral formula I or Ia of the present invention, or a stereoisomer,salt, hydrate, solvate or crystal thereof for prevention or treatment ofviral infection diseases, especially Flaviviridae family viral infectiondiseases, as well as a use of a compound of general formula I or Ia ofthe present invention, or a stereoisomer, salt, hydrate, solvate orcrystal thereof in the manufacture of a medicament for prevention and/ortreatment of viral infection diseases, especially for prevention and/ortreatment of HCV infection diseases, such as HCV viral hepatitis.Examples of such diseases include acute hepatitis C, chronic hepatitisC, and mixed infection of hepatitis C and hepatitis B or hepatitis D.

Definition

Unless otherwise defined, all technical and scientific terms used hereinhave the the same meaning as that commonly understood by a personskilled in the art.

The term “stereoisomer” refers to isomers created by a different spatialarrangement of atoms in molecules, including the cis and trans isomers,and enantiomers and conformational isomers. All stereoisomers are withinthe scope of the present invention. A single stereoisomer of thecompound of the present invention may be substantially free of otherisomers, or mixed into, for example racemates, or mixed with all otherstereoisomers.

The term “salt” refers to a pharmaceutically acceptable salt formed bythe compounds of the present invention with an acid. Said acid may beselected from: phosphoric acid, sulfuric acid, hydrochloric acid,hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid,succinic acid, fumaric acid, acetic acid, lactic acid, nitric acid,sulfonic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acidor and the like.

The term “solvate” refers to a solid or liquid complex formed bycoordination of the compound of the present invention with a solventmolecule. Hydrate is a special form of solvate, wherein the compoundcoordinates with water. In the context of the present invention, hydrateis a preferred solvate.

The term “crystal” refers to all solid forms formed by the compound ofthe present invention, comprising crystalline and amorphous forms.

The term “alkyl” refers to a straight-chain, branched chain or cyclicsaturated hydrocarbon group, preferably with 6 or less carbon atoms.Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl,cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, n-pentyl,isopentyl, neopentyl, cyclohexyl, n-hexyl, isohexyl, 2,2-dimethylbutyland 2,3-dimethylbutyl. The term “C₁₋₆ alkyl” means a straight-chain,branched chain or cyclic saturated hydrocarbon group with 1-6 carbonatoms. The term “C₁₋₄ alkyl” means a straight-chain, branched chain orcyclic saturated hydrocarbon group with 1-4 carbon atoms.

The term “alkenyl” means a straight-chain or branched chain unsaturatedhydrocarbon group containing one or more carbon-carbon double bonds(C═C), preferably with 2 to 6 carbon atoms, more preferably with 2 to 4carbon atoms, and most preferably with 2 carbon atoms. The term “C₂₋₆alkenyl” refers to an unsaturated hydrocarbon group of 2 to 6 carbonatoms containing 1 or 2 carbon-carbon double bonds. The term “C₂₋₄alkenyl” refers to an unsaturated hydrocarbon group of 2 to 4 carbonatoms containing 1 or 2 carbon-carbon double bonds.

The term “alkynyl” means a straight-chain or branched chain unsaturatedhydrocarbon group containing one or more carbon-carbon triple bond(C≡C), preferably with 2 to 6 carbon atoms, more preferably with 2 to 4carbon atoms, most preferably with 2 carbon atoms. The term “C₂₋₆alkynyl” refers to an unsaturated hydrocarbon group of 2 to 6 carbonatoms containing 1 or 2 carbon-carbon triple bonds. The term “C₂₋₄alkynyl” refers to an unsaturated hydrocarbon group of 2 to 4 carbonatoms containing 1 or 2 carbon-carbon triple bonds.

The term “alkoxy” refers to —O— alkyl.

The term “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “halogenated alkyl” means an alkyl substituted by at least onehalogen atom.

The term “heterocyclyl” refers to a cyclic group containing at least oneheteroatom, wherein the heteroatom may be N, O or S, including singleheterocyclyl and fused heterocyclyl. The single heterocyclyl groupincludes, but is not limited to furan, thiophene, pyrrole, thiazole,imidazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-thiadiazole, oxazole,1,2,4-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine,pyrazine, tetrahydrofuran, pyrrolidine, piperidine, piperazine,morpholine, isoxazoline, etc. The fused heterocyclyl group includes, butis not limited to quinoline, isoquinoline, indole, benzofuran,benzothiophene, purine, acridine, carbazole, fluorene, chromene ketone,fluorenone, quinoxaline, 3,4-dihydronaphthalenone, dibenzofuran,dibenzofuran hydride, benzoxazolyl and the like.

The term “benzo five-membered ring” and “benzo six-membered ring” mean afused ring group.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are provided to illustrate the present inventionwithout limiting the invention to the particulars of these examples. Thereagents and starting materials used in the present invention arecommercially obtained.

Example 1:(2S)-2-((([1,1′-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of(2S)-2-((((S)-pentafluorophenoxy)([1,1′-biphenyl]-4-yloxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

To a reaction flask were added 0.9 g phosphorus oxychloride (5.87 mmol)and 30 mL dichloromethane. The mixture was cooled to −60° C., and asolution of g p-phenylphenol (5.87 mmol) and 0.6 g triethylamine (5.87mol) in dichloromethane were added dropwise slowly. After the additionwas complete, the mixture was reacted overnight at room temperature,cooled to 0° C., and 0.9 g L-alanine isopropyl ester hydrochloride (5.3mmol) was added. The mixture was cooled to −60° C., and a solution of1.34 g triethylamine (13 mmol) in dichloromethane (20 mL) was addeddropwise. After the addition was complete, the temperature was raised to−5° C., then a solution of 1 g pentafluorophenol (5.3 mmol) and 0.8 gtriethylamine (8 mmol) in dichloromethane (15 mL) was added dropwise tothe above mixture solution. The resultant mixture was stirred for 1 hourat −5° C. After completion of the reaction, water was added forextraction, then the mixture was dried, concentrated and separated bysilica gel column chromatography to give the title compound.

Step 2: Preparation of(2S)-2-((([1,1′-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

To a reaction flask were added (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine(26 mg, 0.1 mmol) and 1 mL tetrahydrofuran. A solution of 1M tert-butylmagnesium chloride (0.25 mmol) in THF (0.25 mL) was added dropwise undernitrogen protection in an ice bath. After the addition was complete, themixture was reacted for 4 hours at room temperature, and a solution ofthe above-obtained phosphate intermediate of p-phenylpheno (70 mg, 0.13mmol) in tetrahydrofuran (1.5 mL) was added dropwise in an ice bath.After the addition, the mixture was reacted overnight at roomtemperature. After the reaction was complete, the reaction was quenchedby adding 6 mL of 2N HCl in an ice bath. The resultant mixture wasextracted with ethyl acetate, washed with saturated sodium bicarbonatesolution, dried, concentrated, and separated and purified by silica gelcolumn chromatography to give the title compound.

¹HNMR (300 MHz, DMSO) δ: 11.51 (s, 1H, pyrimidineN—H), 7.68-7.62 (m, 4H,Ar—H, pyrimidine-H), 7.60-7.56 (d, 1H, Ar—H), 7.48-7.43 (t, 2H, Ar—H),7.38-7.30 (m, 3H, Ar—H), 6.12-6.00 (m, 2H, tetrahydrofuran-H), 5.84 (d,1H, pyrimidine-H), 5.56 (d, 1H, P—NH), 4.90-4.82 (m, 1H, —(CH3)₂C—H),4.62-4.34 (m, 1H, tetrahydrofuran-OH), 4.30-4.22 (m, 1H, (CH3)C(NH)—H),4.06-4.00 (m, 1H, tetrahydrofuran-H), 3.88-3.77 (m, 2H, P—O—CH₂—H),1.30-1.20 (m, 9H, 3×CH₃), 1.58 (d, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=606.2.

Example 2:(2S)-2-((([1,1′-biphenyl]-3-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using m-phenylphenol, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.50 (s, 1H, pyrimidineN—H), 7.68-7.55 (m, 3H,Ar—H, pyrimidine-H), 7.50-7.36 (m, 6H, Ar—H), 7.21 (d, 1H, Ar—H),6.13-6.00 (m, 2H, tetrahydrofuran-H), 5.88-5.84 (d, 1H, pyrimidine-H),5.57-5.52 (d, 1H, P—NH), 4.86-4.82 (m, 1H, —(CH₃)₂C—H), 4.40-4.38 (m,1H, tetrahydrofuran-OH), 4.28-4.26 (m, 1H, (CH₃)C(NH)—H), 4.04-4.02 (m,1H, tetrahydrofuran-H), 3.88-3.80 (m, 2H, P—O—CH₂—H), 1.28-1.20 (m, 6H,2×CH₃), 1.14-1.11 (d, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=606.2.

Example 3:(2S)-2-((([1,1′-biphenyl]-2-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using o-phenylphenol, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.49 (s, 1H, pyrimidine-N—H), 7.56-7.05 (m,10OH, Ar—H, pyrimidine-H), 6.38-5.89 (m, 2H, tetrahydrofuran-H), 5.80(d, 1H, pyrimidine-H), 5.42 (d, 1H, P—NH), 4.86-4.75 (m, 1H,—(CH₃)₂C—H), 4.24-4.18 (m, 1H, tetrahydrofuran-OH), 4.14-4.08 (m, 1H,(CH₃)C(NH)—H), 3.99-3.89 (m, 1H, tetrahydrofuran-H), 3.85-3.72 (m, 2H,P—O—CH₂—H), 1.27-1.23 (m, 3H, CH₃), 1.19-1.07 (d, 9H, 3×CH₃).

ESI-MS m/z: [M+H]⁺=606.2.

Example 4:(2S)-2-(((2-benzylphen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using o-benzylphenol, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidine-N—H), 7.53-7.05 (m,1OH, Ar—H, pyrimidine-H), 6.36-5.88 (m, 2H, tetrahydrofuran-H), 5.81 (d,1H, pyrimidine-H), 5.43 (d, 1H, P—NH), 4.85-4.71 (m, 1H, —(CH₃)₂C—H),4.21-4.15 (m, 1H, tetrahydrofuran-OH), 4.12-4.01 (m, 1H, (CH₃)C(NH)—H),3.92-3.89 (m, 1H, tetrahydrofuran-H), 3.81-3.73 (m, 2H, P—O—CH₂—H),1.32-1.31 (m, 2H, CH₂) 1.23-1.21 (m, 3H, CH₃), 1.11-1.01 (d, 9H, 3×CH₃).

ESI-MS m/z: [M+H]⁺=620.2.

Example 5:(2S)-2-((([4′-fluoro-1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-fluoro-4′-hydroxybiphenyl, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, CDCl3) δ: 8.09 (s, 1H, pyrimidineN—H), 7.55-7.49 (m, 6H,Ar—H), 7.32 (d 1H, Ar—H), 7.17-7.11 (d, 1H, pyrimidine-H), 6.23-6.18 (m,1H, tetrahydrofuran-H), 5.76 (d, 1H, pyrimidine-H), 5.09-5.00 (m, 1H,P—NH), 4.62-4.49 (m, 2H, —(CH₃)₂C—H), 4.16-4.12 (m, 1H,tetrahydrofuran-OH), 4.04-3.97 (m, 2H, (CH₃)C(NH)—H), 3.78-3.71 (m, 1H,tetrahydrofuran-H), 1.49 (m, 2H, P—O—CH₂—H), 1.42-1.39 (m, 6H, 2×CH₃),1.30-1.24 (d, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=624.3.

Example 6:(2S)-2-((([4′-nitro-1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-4′-nitro biphenyl, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidineN—H), 8.29 (d, 2H,Ar—H), 7.94 (d, 2H, Ar—H), 7.82 (d, 2H, Ar—H), 7.56 (d, 1H,pyrimidine-H), 7.38-7.34 (m, 2H, Ar—H), 6.13-6.04 (m, 2H,tetrahydrofuran-H), 5.83 (d, 1H, pyrimidine-H), 5.56 (d, 1H, P—NH),4.88-4.85 (m, 1H, —(CH₃)₂C—H), 4.40 (m, 1H, tetrahydrofuran-OH), 4.28(m, 1H, (CH3)C(NH)—H), 4.04-4.02 (m, 1H, tetrahydrofuran-H) 3.86-3.84(m, 2H, P—O—CH₂—H), 1.28-1.25 (m, 6H, 2×CH₃), 1.18-1.15 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=651.4.

Example 7:(2S)-2-(((2-methoxy-4-methoxycarbonyl-(E)-ethenylphen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using (E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid methylester, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹HNMR (300 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidine-N—H), 7.62-7.57 (m,2H, Ar—H, pyrimidine-H), 7.46 (s, 1H, CH═CH), 7.31-7.22 (m, 2H, Ar—H),6.63 (d, 1H, CH═CH), 6.03-5.93 (m, 2H, tetrahydrofuran-H), 5.88-5.79 (m,1H, pyrimidine-H), 5.57-5.50 (m, 1H, P—NH), 4.87-4.78 (m, 1H,—(CH3)2C—H), 4.40-4.34 (m, 1H, tetrahydrofuran-OH), 4.25-4.20 (m, 1H,(CH₃)C(NH)—H), 4.05-3.96 (m, 1H, tetrahydrofuran-H), 3.75-3.82 (m, 2H,P—O—CH₂—H), 3.80 (s, 3H, OCH₃), 3.70 (s, 3H, —COOCH₃) 1.26-1.1 (m, 9H,3×CH₃), 1.13-1.10 (m, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=644.2.

Example 8:(2S)-2-((((E)-4-(3,5-dimethoxyphenylethenyl)phen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using (E)-4-(3,5-dimethoxyphenylethenyl)phenol, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.51 (s, 1H, N—H), 8.19 (d, 1H, Ar—H), 7.59(t, 2H, Ar—H), 7.20 (m, 4H, Ar—H), 6.76 (s, 2H, Ar—H), 6.41 (s, 1H,Ar—H), 6.10 (s, 1H, Ar—H), 6.06 (m, 1H, —CH—), 5.85 (d, 1H, N—H), 5.57(d, 1H, —CH—), 4.85 (q, 1H, —CH—), 4.37 (m, 2H, —CH—), 4.27 (m, 1H,—OH), 4.01 (m, 1H, —CH₂—), 3.78 (s, 6H, —CH₃), 1.29 (s, 3H, —CH₃), 1.25(s, 3H, —CH₃), 1.15 (d, 6H, —CH₃).

ESI-MS m/z: [M+H]⁺=692.2.

Example 9:(2S)-2-(((4-methoxycarbonyl-(E)-ethenylphen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using ((E)-3-(4-hydroxyphenyl)acrylic acid methyl ester,phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹HNMR (300 MHz, DMSO) δ: 11.50 (s, 1H, pyrimidine-N—H), 7.63-7.59 (m,2H, Ar—H, pyrimidine-H), 7.48 (s, 1H, CH═CH), 7.32-7.21 (m, 2H, Ar—H),6.65 (d, 1H, CH═CH), 6.01-5.94 (m, 2H, tetrahydrofuran-H), 5.84-5.78 (m,1H, pyrimidine-H), 5.51-5.50 (m, 1H, P—NH), 4.88-4.79 (m, 1H,—(CH₃)₂C—H), 4.42-4.33 (m, 1H, tetrahydrofuran-OH), 4.25-4.18 (m, 1H,(CH₃)C(NH)—H), 4.05-3.95 (m, 1H, tetrahydrofuran-H), 3.75-3.82 (m, 2H,P—O—CH₂—H), 3.72 (s, 3H, —COOCH₃) 1.28-1.12 (m, 9H, 3×CH₃), 1.12-1.10(m, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=644.2.

Example 10:(2S)-2-(((3-cinnamoylphen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 3-cinnamoylphenol, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.52 (s, 1H, pyrimidineN—H), 7.88-7.82 (m, 1H,pyrimidine-H), 7.78-7.72 (m, 3H, Ar—H), 7.60-7.56 (d, 1H, Ar—H), 7.46(s, 1H, CH═CH), 7.31-7.22 (m, 4H, Ar—H), 6.63 (d, 1H, CH═CH), 6.12-6.00(m, 2H, tetrahydrofuran-H), 5.82 (d, 1H, pyrimidine-H), 5.53 (d, 1H,P—NH), 4.91-4.82 (m, 1H, —(CH₃)₂C—H), 4.63-4.34 (m, 1H,tetrahydrofuran-OH), 4.30-4.22 (m, 1H, (CH₃)C(NH)—H), 4.06-4.00 (m, 1H,tetrahydrofuran-H), 3.88-3.77 (m, 2H, P—O—CH₂—H), 1.30-1.20 (m, 9H,3×CH₃), 1.58 (d, 3H, CH₃).

ESI-MS m/z: [M+Na]⁺=682.1.

Example 11:(2S)-2-(((4-(phenylethynyl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 1-(4-hydroxyphenyl)-2-phenyl acetylene, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.49 (s, 1H, pyrimidineN—H), 7.71 (d, 2H,Ar—H), 7.56 (m, 4H, Ar—H, pyrimidine-H), 7.42 (s, 2H, Ar—H), 7.27 (m,1H), 7.04 (m, 1H), 6.16-6.03 (m, 2H, tetrahydrofuran-H), 5.84 (d, 1H,pyrimidine-H), 5.66-5.52 (m, 1H, P—NH), 4.87-4.83 (m, 1H, —(CH₃)₂C—H),4.38 (m, 1H, tetrahydrofuran-OH), 4.25 (m, 1H, (CH3)C(NH)—H), 4.02 (m,1H, tetrahydrofuran-H), 3.79 (m, 2H, P—O—CH2-H), 1.27-1.23 (m, 6H,2×CH₃), 1.16-1.14 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=630.3.

Example 12:(2S)-2-(((4-(1,2,3-thiadiazol-4-yl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(1,2,3-thiadiazol-4-yl)phenol, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.50 (s, 1H, pyrimidineN—H), 9.59 (s, 1H,thiadiazole), 8.14-8.17 (m, 2H, Ar—H), 7.57 (m, 1H, pyrimidine-H),7.36-7.42 (m, 2H, Ar—H), 6.10-6.17 (m, 2H, tetrahydrofuran-H), 5.90 (d,1H, pyrimidine-H), 5.59 (d, 1H, P—NH), 4.90-4.82 (m, 1H, —(CH₃)₂C—H),4.68-4.36 (m, 1H, tetrahydrofuran-OH), 4.31-4.23 (m, 1H, (CH₃)C(NH)—H),4.06-4.02 (m, 1H, tetrahydrofuran-H), 3.89-3.77 (m, 2H, P—O—CH₂—H),1.30-1.20 (m, 9H, 3×CH₃), 1.58 (d, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=614.5.

Example 13:(2S)-2-(((2-(quinoxalin-5-yl)phen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 2-(quinoxalin-5-yl)phenol, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidineN—H), 7.69-7.61 (m, 1H,pyrimidine-H), 7.69-7.61 (m, 3H, Ar—H), 7.61-7.58 (d, 1H, Ar—H),7.49-7.45 (t, 2H, quinoxaline-H), 7.37-7.32 (m, 3H, Ar—H), 6.11-6.03 (m,2H, tetrahydrofuran-H), 5.82 (d, 1H, pyrimidine-H), 5.53 (d, 1H, P—NH),4.90-4.83 (m, 1H, —(CH₃)₂C—H), 4.62-4.32 (m, 1H, tetrahydrofuran-OH),4.30-4.21 (m, 1H, (CH₃)C(NH)—H), 4.04-4.01 (m, 1H, tetrahydrofuran-H),3.83-3.74 (m, 2H, P—O—CH₂—H), 1.34-1.21 (m, 9H, 3×CH₃), 1.53 (d, 3H,CH₃).

ESI-MS m/z: [M+H]⁺=658.2.

Example 14:(2S)-2-(((5,6,7,8-tetrahydronaphthalen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)aceticAcid Methyl Ester

The title compound was prepared according to the method described inExample 1 using 5,6,7,8-tetrahydro-2-naphthol, phosphorus oxychloride,L-amino acetic acid methyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.49 (s, 1H, pyrimidineN—H), 7.58-7.52 (m, 1H,pyrimidine-H), 7.46-7.42 (m, 2H, Ar—H) 7.40-7.36 (d, 1H, Ar—H),6.11-6.00 (m, 2H, tetrahydrofuran-H), 5.82 (d, 1H, pyrimidine-H), 5.55(d, 1H, P—NH), 4.91-4.83 (m, 1H, —(CH3)2C—H), 4.64-4.31 (m, 1H,tetrahydrofuran-OH), 4.31-4.23 (m, 2H, (CH₃)CH₂(NH)), 4.06-4.00 (m, 1H,tetrahydrofuran-H), 3.88-3.77 (m, 2H, P—O—CH₂—H), 1.30-1.20 (m, 3H,CH₃), 1.5 (d, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=542.2.

Example 15:(2S)-2-(((5,6,7,8-tetrahydronaphthalen-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid t-butyl Ester

The title compound was prepared according to the method described inExample 1 using 5,6,7,8-tetrahydro-2-naphthol, phosphorus oxychloride,L-alanine t-butyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.49 (s, 1H, pyrimidineN—H), 7.58-7.52 (m, 1H,pyrimidine-H), 7.46-7.42 (m, 2H, Ar—H) 7.40-7.36 (d, 1H, Ar—H),6.11-6.00 (m, 2H, tetrahydrofuran-H), 5.82 (d, 1H, pyrimidine-H), 5.55(d, 1H, P—NH), 4.64-4.31 (m, 1H, tetrahydrofuran-OH), 4.31-4.23 (m, 1H,(CH₃)C(NH)—H), 4.06-4.00 (m, 1H, tetrahydrofuran-H), 3.88-3.77 (m, 2H,P—O—CH₂—H), 1.33-1.31 (m, 8H, 4×CH₂) 1.30-1.20 (m, 12H, 4×CH₃), 1.5 (d,3H, CH₃).

ESI-MS m/z: [M−H]⁺=596.3.

Example 16:(2S)-2-(((4-(thiazol-2-yl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 2-(4-methoxyphenyl)thiazole

To a 100 mL single-neck flask were added 2.2 g p-methoxyphenyl boronicacid (0.014 mol), 2 g 4-bromothiazole (0.012 mol), 7.8 g cesiumcarbonate (0.024 mol) and 0.4 g bis(triphenylphosphine)palladiumdichloride (0.6 mmol), then added 30 mL 1,4-dioxane and 8 mL water. Themixture was heated to 90° C. under nitrogen protection, and was reactedovernight. The reaction was stopped after the starting materials werealmost reacted completely. The mixture was cooled to room temperature,concentrated, and extracted with water and ethyl acetate. The organicphases were combined, dried, concentrated, and separated by silica gelcolumn chromatography to give the title compound.

¹HNMR (300 MHz, CDCl3) δ: 7.90-7.95 (m, 2H, Ar—H), 7.83 (d, 1H, Ar—H),7.26 (d, 1H, Ar—H), 6.95-7.00 (m, 2H, Ar—H), 3.88 (s, 3H, OCH₃).

ESI-MS m/z: [M+H]⁺=192.2.

Step 2: Preparation of 4-(thiazol-2-yl)phenol

To a 100 mL single-neck flask were added the product obtained from Step1 (0.9 g, 4.7 mmol) and 40% HBr. The product from Step 1 was completelydissolved, and the resulting solution was yellow transparent. 6 mLacetic acid was added, and the solution was heated to 115° C. andreacted for 20 hours. The reaction was stopped after the startingmaterials almost disappeared. The mixture was concentrated to drynessand separated by silica gel column chromatography to give 0.42 g of apale yellow solid.

¹HNMR (300 MHz, CDCl3) δ: 7.83-7.88 (m, 3H, Ar—H), 7.30 (d, 1H, Ar—H),6.92 (d, 2H, Ar—H).

ESI-MS m/z: [M+H]⁺=178.2.

Step 3 Preparation of(2S)-2-(((4-(thiazol-2-yl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(thiazol-2-yl)phenol, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.47 (s, 1H, pyrimidineN—H), 7.97-7.90 (m, 3H,Ar-2H, thiazole-1H), 7.76 (d, 1H, thiazole-H), 7.56 (d, 1H,pyrimidine-H), 7.35 (d, 2H, Ar—H), 6.12-6.04 (m, 2H, tetrahydrofuran-H),5.82 (d, 1H, pyrimidine-H), 5.58-5.56 (m, 1H, P—NH), 4.88-4.83 (m, 1H,—(CH₃)₂C—H), 4.39 (m, 1H, tetrahydrofuran-OH), 4.28-4.27 (m, 1H,(CH₃)C(NH)—H), 4.05-4.01 (m, 1H, tetrahydrofuran-H), 3.84-3.83 (m, 2H,P—O—CH₂—H), 1.28-1.24 (m, 6H, 2×CH₃), 1.16-1.14 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=613.3.

Example 17:(2S)-2-(((4-(thiazol-5-yl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 5-(4-methoxyphenyl)thiazole

To a 100 mL single-neck flask were added 2.2 g p-methoxyphenylboronicacid (0.014 mol), 2 g 5-bromothiazole (0.012 mol), 7.8 g cesiumcarbonate (0.024 mol) and 0.4 g bis(triphenylphosphine)palladiumdichloride (0.6 mmol), then added 30 mL 1,4-dioxane and 8 mL water. Themixture was heated to 90° C. under nitrogen protection, and was reactedovernight. The reaction was stopped after the starting materials werealmost reacted completely. The mixture was cooled to room temperature,concentrated, and extracted with water and ethyl acetate. The organicphases were combined, dried, concentrated, and separated by silica gelcolumn chromatography to give a pale yellow solid product.

¹HNMR (300 MHz, CDCl₃) δ: 8.74 (s, 1H, Ar—H), 8.00 (s, 1H, Ar—H),7.51-7.54 (m, 2H, Ar—H), 6.95-6.99 (m, 2H, Ar—H), 3.87 (s, 3H, OCH₃).

ESI-MS m/z: [M+H]⁺=192.2.

Step 2: Preparation of 4-(thiazol-5-yl)phenol

To a 100 mL single-neck flask were added the product obtained from Step1 (0.9 g, 4.7 mmol) and 40% HBr. The product from Step 1 was completelydissolved, and the resulting solution was yellow transparent. 6 mLacetic acid was added, and the mixture was heated to 115° C. and reactedfor 20 hours. The reaction was stopped after the starting materialalmost disappeared. The mixture was concentrated to dryness, extractedwith water and ethyl acetate, dried, concentrated, and separated bysilica gel column chromatography to give 0.66 g of a pale yellow solid.

¹HNMR (300 MHz, CDCl₃) δ: 8.72 (s, 1H, Ar—H), 7.99 (s, 1H, Ar—H),7.43-7.49 (m, 2H, Ar—H), 6.87-6.90 (m, 2H, Ar—H).

ESI-MS m/z: [M+H]⁺=178.2.

Step 3: Preparation of(2S)-2-(((4-(thiazol-5-yl)phen-1-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(thiazol-5-yl)phenol, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidineN—H), 9.06 (s, 1H,thiazole-H), 7.97 (d, 1H, thiazole-1H), 7.70 (d, 2H, Ar—H), 7.56 (d, 1H,pyrimidine-H), 7.31 (d, 2H, Ar—H), 6.09-6.01 (m, 2H, tetrahydrofuran-H),5.81 (d, 1H, pyrimidine-H), 5.57-5.56 (m, 1H, P—NH), 4.88-4.83 (m, 1H,—(CH₃)₂C—H), 4.38 (m, 1H, tetrahydrofuran-OH), 4.28-4.27 (m, 1H,(CH₃)C(NH)—H), 4.05-4.02 (m, 1H, tetrahydrofuran-H), 3.85-3.83 (m, 2H,P—O—CH₂—H), 1.28-1.23 (m, 6H, 2×CH₃), 1.18-1.14 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=613.4.

Example 18:(2S)-2-((([4′-cyano-1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4′-cyano-4-hydroxybiphenyl, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

1HNMR (300 MHz, DMSO) δ: 11.50 (s, 1H, pyrimidineN—H), 7.98-7.82 (m, 8H,Ar—H), 7.57 (m, 1H, pyrimidine-H), 6.18-6.15 (m, 2H, tetrahydrofuran-H),5.91 (d, 1H, pyrimidine-H), 5.59 (d, 1H, P—NH), 4.92-4.87 (m, 1H,—(CH₃)₂C—H), 4.68-4.36 (m, 2H, tetrahydrofuran-OH, (CH₃)C(NH)—H),4.03-4.01 (m, 1H, tetrahydrofuran-H), 3.85-3.82 (m, 2H, P—O—CH₂—H),1.40-1.29 (m, 9H, 3×CH₃), 1.15-1.13 (d, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=631.2.

Example 19:(2S)-2-(((4-((E)-4-fluorophenylethenylphenyl)-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of (E)-4-(4-fluorophenylethenyl)phenol

To a 50 mL three-necked flask were added p-iodophenol (1.32 g, 6 mmol),p-fluorostyrene (610 mg, 5 mmol), tetrakis(triphenylphosphine)palladium(622 mg, 0.5 mmol) and cesium carbonate (4.89 g, 15 mmol), then added 20mL of 1,4-dioxane. Argon gas was passed through the solution, and themixture was reacted at 80° C. overnight (about 10 h) until the solutionturned black. After 24 h, the reaction was complete by TLC monitoring.Dilute hydrochloric acid was slowly added dropwise at 0° C., and themixture was extracted with ethyl acetate and separated by silica gelcolumn chromatography to give a white solid.

ESI-MS m/z: [M+H]⁺=215.2.

Step 2: Preparation of(2S)-2-((((E)-4-(4-fluorophenylethenylphenyl)-1-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using (E)-4-(4-fluorophenylethenyl)phenol, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.46 (s, 1H, pyrimidineN—H), 7.64-7.58 (m, 5H,Ar—H, pyrimidine-H), 7.23-7.15 (m, 6H, Ar—H, —CH═CH—), 6.10-6.15 (m, 2H,tetrahydrofuran-H), 5.91 (d, 1H, pyrimidine-H), 5.79 (d, 1H, P—NH),4.91-4.88 (m, 1H, —(CH₃)₂C—H), 4.66-4.32 (m, 2H, tetrahydrofuran-OH,(CH₃)C(NH)—H), 4.03-4.01 (m, 1H, tetrahydrofuran-H), 3.85-3.82 (m, 2H,P—O—CH₂—H), 1.40-1.29 (m, 9H, 3×CH₃), 1.15-1.13 (d, 3H, CH₃).

ESI-MS m/z: [M+H]⁺=650.4.

Example 20:(2S)-2-(((1H-indol-5-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 5-hydroxy-1H-indole, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.54 (s, 1H, pyrimidine-N—H), 11.14 (s, 1H,Indole-N—H), 7.55 (d, 1H, pyrimidine-H), 7.37-7.32 (m, 3H, Indole-H),6.96 (d, 1H, Indole-H), 6.38 (s, 1H, Indole-H), 6.05-5.84 (m, 3H,tetrahydrofuran-H, pyrimidine-H), 5.55-5.48 (m, 1H, P—NH), 4.88-4.80 (m,1H, —(CH₃)₂C—H), 4.38-4.33 (m, 1H, tetrahydrofuran-OH), 4.25-4.20 (m,1H, (CH₃)C(NH)—H), 4.03-4.00 (m, 1H, tetrahydrofuran-H), 3.82-3.74 (m,2H, P—O—CH₂—H), 1.28-1.21 (m, 3H, CH₃), 1.17-1.15 (m, 3H, CH₃),1.15-1.12 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=569.2.

Example 21:(2S)-2-(((4-fluoro-2-methyl-1H-indol-5-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-fluoro-5-hydroxy-2-methyl-1H-indole, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidine-N—H), 11.19 (s, 1H,Indole-N—H), 7.55 (d, 1H, pyrimidine-H), 7.02 (s, 2H, Ar—H), 6.17 (d,1H, Ar—H), 5.97-5.85 (m, 3H, tetrahydrofuran-H, pyrimidine-H), 5.56-5.49(m, 1H, P—NH), 4.86 (m, 1H, —(CH₃)₂C—H), 4.41-4.35 (m, 1H,tetrahydrofuran-OH), 4.25-4.20 (m, 1H, (CH₃)C(NH)—H), 4.04-4.00 (m, 1H,tetrahydrofuran-H), 3.82-3.78 (m, 2H, P—O—CH₂—H), 2.37 (s, 3H, CH₃),1.25-1.22 (m, 6H, 2×CH₃), 1.16-1.14 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=601.3.

Example 22:(2S)-2-(((3-methoxycarbonylmethyl-benzofuran-7-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 7-hydroxy-3-methoxycarbonylmethylbenzofuran, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.48 (s, 1H, pyrimidine-N—H), 7.95 (s, 1H,Furan-H), 7.56 (d, 1H, pyrimidine-H), 7.40 (d, 1H, Ar—H), 7.28 (s, 1H,Ar—H), 7.20 (t, 1H, Ar—H), 6.13 (m, 1H, tetrahydrofuran-H), 6.01 (m, 1H,pyrimidine-H), 5.82 (m, 1H, tetrahydrofuran-H), 5.50 (m, 1H, P—NH), 4.86(m, 1H, —(CH₃)₂C—H), 4.42 (m, 1H, tetrahydrofuran-OH), 4.31 (m, 1H,(CH₃)C(NH)—H), 4.02 (m, 1H, tetrahydrofuran-H), 3.84-3.82 (m, 4H,P—O—CH₂—H, —CH₂—), 3.65 (s, 3H, CH₃), 1.27-1.22 (m, 6H, 2×CH₃),1.16-1.14 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+H]⁺=642.2.

Example 23:(2S)-2-(((4-oxo-2-phenyl-4H-benzopyran-6-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 6-hydroxyflavone, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 13.22 (s, 1H, N—H), 8.59 (s, 1H, Ar—H), 8.50(d, 2H, Ar—H), 8.30 (s, 1H, Ar—H), 8.20 (d, 1H, Ar—H), 8.03 (d, 1H,Ar—H), 7.70 (dd, 2H, Ar—H), 7.61 (d, 1H, Ar—H), 7.48 (m, 1H, Ar—H), 7.38(d, 1H, Ar—H), 7.36 (d, 1H, N—H), 7.34 (d, 1H, —CH—), 7.26 (m, 1H,—CH—), 5.62 (s, 2H, —CH₂—), 5.54 (s, 1H, —OH), 5.32 (m, 1H, —CH—), 4.59(m, 1H, —CH—), 4.05 (m, 1H, —CH—), 2.01 (s, 3H, —CH₃), 1.35 (s, 3H,—CH₃), 1.16 (d, 6H, —CH₃).

ESI-MS m/z: [M+H]⁺=674.2.

Example 24:(2S)-2-(((quinoxalin-5-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 5-hydroxyquinoxaline, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.52 (s, 1H, pyrimidine-N—H), 7.54 (d, 1H,pyrimidine-H), 7.36-7.31 (m, 3H, Ar—H), 6.95 (d, 1H, Ar—H), 6.36 (d, 1H,Ar—H), 6.03-5.82 (m, 3H, tetrahydrofuran-H, pyrimidine-H), 5.55-5.48 (m,1H, P—NH), 4.88-4.80 (m, 1H, —(CH₃)₂C—H), 4.38-4.33 (m, 1H,tetrahydrofuran-OH), 4.25-4.20 (m, 1H, (CH₃)C(NH)—H), 4.03-4.01 (m, 1H,tetrahydrofuran-H), 3.82-3.74 (m, 2H, P—O—CH₂—H), 1.27-1.25 (m, 3H,CH₃), 1.18-1.16 (m, 3H, CH₃), 1.15-1.12 (m, 6H, 2×CH₃).

ESI-MS m/z: [M+Na]⁺=604.1.

Example 25:(2S)-2-(((4-oxo-2-phenylbenzodihydropyran-7-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 7-hydroxyflavanone, phosphorus oxychloride, L-alanineisopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (300 MHz, DMSO) δ: 11.59 (s, 1H, pyrimidineN—H), 8.06-8.02 (m, 1H,Ar—H), 7.89-7.86 (m, 3H, Ar—H) 7.69-7.61 (m, 1H, pyrimidine-H),7.42-7.41 (m, 2H, Ar—H), 7.36-7.34 (d, 2H, Ar—H), 6.12-6.03 (m, 2H,tetrahydrofuran-H), 5.84 (d, 1H, pyrimidine-H), 5.43 (d, 1H, P—NH),4.90-4.83 (m, 1H, —(CH₃)₂C—H), 4.63-4.32 (m, 1H, tetrahydrofuran-OH),4.30-4.21 (m, 1H, (CH₃)C(NH)—H), 4.14-4.11 (m, 1H, tetrahydrofuran-H),4.10-4.08 (m, 2H, P—O—CH₂—H), 3.98-3.96 (m, 4H, CH₂) 1.98-1.95 (m, 1H,CH) 1.34-1.21 (m, 6H, 2×CH₃), 1.53 (d, 6H, 2×CH₃) ESI-MS m/z:[M+H]⁺=676.2.

Example 26: (2S)-2-((((4-(1H-1,2,4-triazol-1-yl)phenyloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(1H-1,2,4-triazol-1-yl)phenol, phosphorus oxychloride,L-alanine isopropyl ester hydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹H NMR (300 MHz, DMSO-d₆) δ ppm: 11.48 (s, 1H), 9.18-9.21 (m, 2H),8.15-8.18 (m, 1H), 8.05-8.07 (m, 1H), 7.98-8.02 (m, 1H), 7.56-7.58 (m,1H), 7.15-7.21 (m, 1H), 6.11-6.15 (m, 2H), 5.92 (m, 1H), 5.88 (m, 1H),4.88-4.92 (m, 1H), 4.32-4.65 (m, 2H), 4.01-4.04 (m, 1H), 3.82-3.86 (m,2H), 1.13-1.40 (m, 12H).

LC-MS m/z: [M+H]⁺=597.

Example 27:(2S)-2-((((4′-chloro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4′-chloro-4-hydroxy[1,1′-biphenyl], phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹H NMR (300 MHz, DMSO-d₆) δ ppm: 11.54 (s, 1H), 7.66-7.69 (d, 3H),7.55-7.59 (d, 1H), 7.49-7.52 (d, 2H), 7.29-7.33 (m, 3H), 6.12-6.16 (m,2H), 5.96-5.99 (m, 1H), 5.34 (m, 1H), 4.81-4.89 (m, 1H), 4.38-4.42 (m,2H), 3.84 (m, 1H), 3.82-3.84 (m, 2H), 1.05-1.14 (m, 12H).

LC-MS m/z [M+H]⁺=640.

Example 28:(2S)-2-(((2-fluoro-[1,1′-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 2-fluoro-4-hydroxy-[1,1′-biphenyl]

In a 250 mL egg plant-shaped flask were added 1.06 g phenylboronic acid,2.38 g 3-fluoro-4-iodophenol, 0.4 g Pd(dppf)Cl₂ and 9 g cesiumcarbonate, then added 50 mL 1,4-dioxane and 5 mL water. The mixture wasstirred at 90° C. for 1.5 h under nitrogen protection, then the reactionwas stopped. The reaction solution was extracted with 100 mL ethylacetate and 50 mL saturated sodium chloride solution, and washed withwater (3×50 mL). The organic phases were collected, dried over anhydroussodium sulfate, filtered, concentrated, and purified by columnchromatography to give the title compound.

LC-MS m/z: [M+H]⁺=189.

Step 2: Preparation of(2S)-2-(((2-fluoro-[1,1′-biphenyl]-4-yloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 2-fluoro-4-hydroxy-[1,1′-biphenyl]prepared from Step 1,phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.49 (s, 1H), 7.51-7.82 (m, 6H),7.15-7.29 (m, 3H), 6.12-6.15 (m, 1H), 6.03 (m, 1H), 5.97-5.99 (m, 1H),5.58 (m, 1H), 4.80-4.89 (m, 1H), 4.38-4.41 (m, 2H), 4.01 (m, 1H),3.82-3.84 (m, 2H), 1.05-1.14 (m, 12H).

LC-MS m/z: [M+H]⁺=624.

Example 29:(2S)-2-(((4-(pyrimidin-2-yl)phenyloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 2-(4-methoxyphenyl)pyrimidine

The title compound was prepared according to the method described inStep 1 of Example 28 using 4-methoxyphenylboronic acid,2-bromopyrimidine, Pd(dppf)Cl₂ and cesium carbonate as startingmaterials.

LC-MS m/z: [M+H]⁺=187.

Step 2: Preparation of 4-(pyrimidin-2-yl)phenol

To a 50 mL round-bottomed flask was added 830 mg2-(4-methoxyphenyl)pyrimidine prepared from Step 1 in dichloromethane(10 mL). 8 mL of 1N BBr₃ was slowly added dropwise at −20° C. undernitrogen protection. After the addition was complete, the mixture wasstirred at 0° C. After the reaction was complete, the mixture wasquenched by adding 10 mL water, and extracted with dichloromethane (2×20mL). The organic phases were collected, washed with water and saturatedbrine, dried over anhydrous sodium sulfate, filtered, concentrated, andpurified by column chromatography to give the title compound.

LC-MS m/z: [M+H]⁺=173.

Step 3: Preparation of(2S)-2-(((4-(pyrimidin-2-yl)phenyloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(pyrimidin-2-yl)phenol prepared from Step 2,phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹HNMR (500 MHz, DMSO-d₆) δppm: 11.48 (s, 1H), 8.88-8.89 (m, 2H),8.38-8.40 (m, 2H), 7.57-7.59 (m, 1H), 7.43-7.44 (m, 1H), 7.35-7.28 (m,2H), 6.13-6.15 (m, 1H), 6.04 (m, 1H), 5.86-5.88 (m, 1H), 5.59 (m, 1H),4.81-4.89 (m, 1H), 4.38-4.41 (m, 2H), 4.02 (m, 1H), 3.81-3.84 (m, 2H),1.10-1.18 (m, 12H).

LC-MS m/z: [M+Na]⁺=630.

Example 30:(2S)-2-((((3′-(N-methylcarbamoyl)-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: 4′-hydroxy-N-methyl-[1,1′-biphenyl]-3-formamide

The title compound was prepared according to the method described inStep 1 of Example 28 using 4-iodophenol,3-(N-methylcarbamoyl)phenylboronic acid, Pd(dppf)Cl₂ and cesiumcarbonate as starting materials.

LC-MS m/z: [M+H]⁺=228.

Step 2: Preparation of(2S)-2-((((3′-(N-methylcarbamoyl)-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4′-hydroxy-N-methyl-[1,1′-biphenyl]-3-formamide preparedfrom Step 1, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.46-11.50 (s, 1H), 7.95-7.96 (m, 1H),7.70-7.73 (m, 1H), 7.57-7.59 (m, 1H), 7.35-7.37 (m, 2H), 7.30-7.32 (m,1H), 7.28-7.31 (m, 1H), 7.22-7.26 (m, 1H), 7.18-7.20 (m, 1H), 7.15-7.17(m, 1H), 6.08-6.10 (m, 1H), 6.01 (m, 1H), 5.97-6.02 (m, 1H), 5.58 (m,1H), 4.81-4.88 (m, 1H), 4.38-4.42 (m, 2H), 4.05 (m, 1H), 3.89-3.92 (m,5H), 1.16-1.28 (m, 12H).

LC-MS m/z: [M−H]⁺=661.

Example 31:(2S)-2-((((3′-fluoro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-(3-fluorophenyl)phenol

The title compound was prepared according to the method described inStep 1 of Example 28 using 4-hydroxyphenylboronic acid,3-fluoroiodobenzene, Pd(dppf)Cl₂ and cesium carbonate as startingmaterials.

LC-MS m/z: [M+H]⁺=189.

Step 2: Preparation of(2S)-2-((((3′-fluoro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(3-fluorophenyl)phenol prepared from Step 1,phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.49 (s, 1H), 7.95-7.97 (m, 1H),7.70-7.72 (m, 1H), 7.56-7.58 (m, 1H), 7.36-7.38 (m, 2H), 7.31-7.32 (m,1H), 7.28-7.30 (m, 1H), 7.19-7.20 (m, 1H), 7.16-7.17 (m, 1H), 6.07-6.10(m, 1H), 6.03 (m, 1H), 5.97-6.01 (m, 1H), 5.56 (m, 1H), 4.80-4.88 (m,1H), 4.38-4.41 (m, 2H), 4.06 (m, 1H), 3.82-3.86 (m, 2H), 1.16-1.27 (m,12H).

LC-MS m/z: [M+H]⁺=624.

Example 32:(2S)-2-((((3′-chloro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-(3-chlorophenyl)phenol

The title compound was prepared according to the method described inStep 1 of Example 28 using 4-hydroxyphenylboronic acid,3-chloroiodobenzene, Pd(dppf)Cl₂ and cesium carbonate as startingmaterials.

LC-MS m/z: [M+H]⁺=205.

Step 2: Preparation of(2S)-2-((((3′-chloro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(3-chlorophenyl)phenol prepared from Step 1,phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.49 (s, 1H), 7.51-7.82 (m, 6H),7.26-7.35 (m, 3H), 6.12-6.14 (m, 1H), 6.11 (m, 1H), 5.96-5.98 (m, 1H),5.88 (m, 1H), 4.80-4.88 (m, 1H), 4.38-4.42 (m, 2H), 4.01 (m, 1H),3.82-3.87 (m, 2H), 1.11-1.29 (m, 12H).

LC-MS m/z: [M+H]⁺=640.

Example 33:(2S)-2-(((S)-((4-fluoro-1,2-dimethyl-1H-indol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 2-(2,3-difluoro-6-nitrophenyl)-3-oxobutanoic acidethyl ester

To a 100 mL round-bottomed flask was added 35 mL tetrahydrofuran, slowlyadded 1150 mg sodium hydride with stirring in an ice bath, and slowlyadded 1.43 mL ethyl acetoacetate. After the addition was complete, asolution of 2.3 mL 2,3,4-trifluoronitrobenzene in tetrahydrofuran (4 mL)was added slowly. After the addition was complete, the mixture wasreacted for 24 hours at room temperature, then the reaction was stoppedby water quenching. 35 mL 2N HCl was added to adjust the pH to neutral,and the mixture was extracted with ethyl acetate. The organic layerswere collected, dried over anhydrous sodium sulfate, filtered, andconcentrated to give the title compound, which was used directly in thenext step.

Step 2: Preparation of 1-(2,3-difluoro-6-nitrophenyl)propan-2-one

2-(2,3-difluoro-6-nitrophenyl)-3-oxobutanoic acid ethyl ester preparedfrom Step 1 was transferred to a reaction flask, and a mixture of 10 mLconcentrated hydrochloric acid and 10 mL glacial acetic acid was added.The solution was reacted for 12 h at 100° C. before the reaction wasstopped. The solution was diluted with water and ethyl acetate, and theethyl acetate layers were obtained. Saturated aqueous NaHCO₃ solutionwas added to adjust the pH to neutral, the ethyl acetate layer wasseparated, and the aqueous phase was extracted with ethyl acetate. Theorganic layers were combined, dried over anhydrous Na₂SO₄, filtered,concentrated, and purified by column chromatography to give the titlecompound.

¹HNMR (400 MHz, DMSO-d₆) δppm: 8.04-8.08 (m, 1H), 7.66-7.7 (m, 1H),4.26-4.26 (d, 2H), 2.30 (s, 3H).

Step 3: Preparation of 1-(2-fluoro-3-methoxy-6-nitrophenyl)propan-2-one

In a 100 mL round-bottomed flask 2 g1-(2,3-difluoro-6-nitrophenyl)propan-2-one prepared from Step 2 wasadded to a solution of sodium methoxide in methanol (40 mL), wherein thesolution of sodium methoxide in methanol was prepared by slowly adding2.27 g Na to methanol with stirring in an ice bath and stirring for 1 hat 50° C. after the addition. The mixture was reacted for 1 h at roomtemperature before the reaction was complete, and quenched by adding 1mL water and extracted with dichloromethane. The organic layers werewashed with saturated brine, dried over anhydrous sodium sulfate,filtered, and concentrated to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.04-8.07 (m, 1H), 7.31-7.35 (m, 1H),4.20-4.21 (d, 2H), 3.98 (s, 3H), 2.28 (s, 3H).

Step 4: Preparation of 2-methyl-4-fluoro-5-methoxy-1H-indole

In a 100 mL round-bottomed flask 1.9 g1-(2-fluoro-3-methoxy-6-nitrophenyl)propan-2-one prepared from Step 3was added to 15 mL methanol and dissolved, and 190 mg palladium oncarbon was added. The mixture was reacted for 36 h at room temperature,then the reaction was stopped. The mixture was filtered, andconcentrated to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 11.01 (br, 1H), 6.99-7.01 (d, 1H),6.853 (m, 1H), 6.10-6.11 (t, 1H), 3.79 (s, 3H), 2.35 (s, 3H).

Step 5: Preparation of 1,2-dimethyl-4-fluoro-5-methoxy-1H-indole

In a 100 mL round-bottomed flask 950 mg2-methyl-4-fluoro-5-methoxy-1H-indole prepared from Step 4 was dissolvedin 6 mL tetrahydrofuran, and 87.2 mg sodium hydride was slowly added at0° C. After the addition was complete, the solution was kept stirringfor 30 min, and 0.14 mL iodomethane was slowly added at 0° C. Themixture was reacted for 1 h before the reaction was stopped, thenquenched by slowly adding water at 0° C., extracted with ethyl acetate,washed with saturated brine, dried over anhydrous sodium sulfate,filtered, concentrated, and purified by column chromatography to givethe title compound.

Step 6: Preparation of 1,2-dimethyl-4-fluoro-5-hydroxy-1H-indole

In a 100 mL round-bottomed flask 210 mg1,2-dimethyl-4-fluoro-5-methoxy-1H-indole prepared from Step 5 wasdissolved in 4 mL anhydrous dichloromethane, and 0.2 mL BBr₃ was slowlyadded at −70° C. The reaction was kept for 2 h at −70° C. before thereaction was stopped. The reaction solution was slowly poured into icewater, and the dichloromethane was removed under reduced pressure. Theresidue was filtered, and the filter cake was dissolved in methanol, andconcentrated to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ: 8.82 (s, 1H), 6.97-6.99 (d, 1H), 6.68-6.72(t, 1H), 8.12 (s, 1H), 3.59 (s, 3H), 2.35 (s, 3H).

Step 7(2S)-2-(((S)-((4-fluoro-1,2-dimethyl-1H-indol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 1,2-dimethyl-4-fluoro-5-hydroxy-1H-indole prepared fromStep 6, phosphorus oxychloride, L-alanine isopropyl ester hydrochloride,pentafluorophenol and (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine asstarting materials.

¹HNMR (400 MHz, DMSO-d₆) δ: 11.48 (s, 1H), 7.52-7.54 (d, 1H), 7.08-7.19(m, 2H), 6.29 (s, 1H), 5.86-6.08 (m, 3H), 5.47-5.49 (d, 1H), 4.84-4.87(t, 1H), 4.24-4.36 (m, 2H), 3.77-3.86 (m, 3H), 3.65 (s, 3H), 2.40 (s,3H), 1.21-1.27 (m, 6H), 1.14-1.16 (m, 6H).

LC-MS m/z: [M+H]⁺=615.

Example 34:(2S)-2-((((6-fluoro-3-methylbenzo[d]isoxazol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 3-fluoro-4-methoxybenzaldehyde

In a 100 mL round-bottomed flask 1.3 mL 3,4-difluorobenzaldehyde wasdissolved in 30 mL methanol, and 2.2 g sodium methoxide was added slowlyat room temperature. The mixture was reacted for 12 h before thereaction was stopped, and quenched by adding 3 mL water. Methanol wasremoved under reduced pressure, and the residue was extracted withdichloromethane. The organic phases were collected, washed withsaturated brine, dried over anhydrous sodium sulfate, filtered,concentrated, and purified by column chromatography to give the titlecompound.

¹HNMR (400 MHz, DMSO-d₆) δppm: 9.87-9.88 (d, 1H), 7.81-7.82 (m, 1H),7.79 (m, 1H), 7.40-7.42 (t, 1H), 3.96 (s, 3H).

Step 2: Preparation of (3-fluoro-4-methoxyphenyl)formate

In a 100 mL round-bottomed flask 2.5 g 3-fluoro-4-methoxybenzaldehydeprepared from Step 1 was dissolved in 20 mL anhydrous dichloromethane,and 4.7 g m-chloroperbenzoic acid was added. The mixture was reacted for4 h at 50° C. before the reaction was stopped. The mixture was dilutedwith 30 mL dichloromethane, washed with water, brine, dried overanhydrous sodium sulfate, filtered, and concentrated to give the titlecompound, which was used directly in the next step.

Step 3: Preparation of 3-fluoro-4-methoxyphenol

To a 100 mL round-bottomed flask was added 2.4 g(3-fluoro-4-methoxyphenyl)formate prepared from Step 2, and added 10 mL5% sodium hydroxide solution and 20 mL methanol. The mixture was reactedfor 1 h at room temperature before the reaction was stopped. 2Nhydrochloric acid was added to adjust the pH to about 5, and methanolwas removed under reduced pressure. The residue was extracted with ethylacetate, and washed with water and saturated brine. The organic phaseswere collected, dried over anhydrous sodium sulfate, filtered,concentrated, and purified by column chromatography to give the titlecompound.

¹HNMR (400 MHz, DMSO-d₆) δppm: 9.36 (s, 1H), 6.93-6.98 (m, 1H),6.59-6.62 (m, 1H), 6.50-6.51 (m, 1H), 3.73 (s, 3H).

Step 4: Preparation of (3-fluoro-4-methoxyphenyl)acetate

1.8 g 3-fluoro-4-methoxyphenol prepared from Step 3 was added to a 100mL round-bottomed flask, and dissolved by adding 25 mL dichloromethane.1 mL acetyl chloride and 1.9 mL triethylamine were added in an ice bath,and the mixture was reacted for 10 min before the reaction was stopped.40 mL diethyl ether was added, and the reaction solution was filtered.The filtrate was dried by rotary evaporation to give a yellow solid, towhich 30 mL diethyl ether was added. The solution was filtered, and thefiltrate was dried by rotary evaporation to give the title compound,which was used directly in the next step.

Step 5: Preparation of 1-(4-fluoro-2-hydroxy-5-methoxyphenyl)ethanone

2 g (3-fluoro-4-methoxyphenyl)acetate prepared from Step 4 was added toa 100 mL round-bottomed flask, and 6.5 mL trifluoromethanesulfonic acidwas slowly added at 0° C. The mixture was stirred for another 5 min, andreacted for 75 min at 70° C. before the reaction was stopped. Themixture was cooled to 0° C., and 20 mL ice water was added. The reactionsolution was filtered, and the filter cake was washed with water. Thefilter cake was collected, and dried in vacuum to give the titlecompound.

¹HNMR (400 MHz, DMSO-d₆) δppm: 11.95-11.95 (d, 1H), 7.51-7.54 (d, 1H),6.89-6.92 (d, 1H), 3.85 (s, 3H), 2.65 (s, 3H).

LC-MS m/z: [M+H]⁺=185.

Step 6: Preparation of (E)-1-(4-fluoro-2-hydroxy-5-methoxyphenyl)ethylketoxime

2.4 g 1-(4-fluoro-2-hydroxy-5-methoxyphenyl)ethanone prepared from Step5 and 1.08 g hydroxylamine hydrochloride were added to a 50 mLround-bottomed flask and dissolved by adding 15 mL ethanol, and 0.6 gsodium hydroxide was slowly added at 50° C. After the addition wascomplete, the mixture was refluxed for about 1 h before the reaction wasstopped. The reaction solution was vigorously stirred at 0-4° C., and 20mL water was added. The reaction solution was filtered, and the filtercake was washed with cold water, and dried in vacuum to give the titlecompound.

¹HNMR (400 MHz, DMSO-d₆) δppm: 11.53 (s, 1H), 11.49 (s, 1H), 7.14-7.17(d, 1H), 6.75-6.782 (d, 1H), 3.81 (s, 3H), 2.23 (s, 3H).

LC-MS m/z: [M+H]⁺=200.

Step 7: Preparation of 6-fluoro-5-methoxy-3-methylbenzo[d]isoxazole

800 mg (E)-1-(4-fluoro-2-hydroxy-5-methoxyphenyl)ethyl ketoxime preparedfrom Step 6 and 332 mg potassium carbonate were added to a 25 mLround-bottomed flask. 5.8 mL N-methylpyrrolidone (NMP) and 0.4 mL aceticanhydride were added. The mixture was reacted for 3 h at 120° C. undernitrogen protection before the reaction was stopped. The reactionsolution was cooled to room temperature, and 10 mL water was added. Thereaction solution was filtered, and the filter cake was dissolved withdichloromethane, and dried over anhydrous sodium sulfate, filtered, andconcentrated to give the title compound.

¹HNMR (400 MHz, DMSO-d₆) δ ppm: 7.73-7.76 (d, 1H), 7.55-7.57 (d, 1H),3.39 (s, 3H), 2.53 (s, 3H).

LC-MS m/z: [M+H]⁺=182.

Step 8: Preparation of 3-methyl-5-hydroxy-6-fluoro-benzo[d]isoxazole

400 mg 6-fluoro-5-methoxy-3-methylbenzo[d]isoxazole prepared from Step 7was added to a 25 mL round-bottomed flask and dissolved by adding 6 mLanhydrous dichloromethane. 0.5 mL boron tribromide was added dropwiseslowly at −70° C. under nitrogen protection. After the addition wascomplete, the mixture was reacted for another 30 min, then the reactionwas stopped. The reaction solution was poured into crushed ice, anddichloromethane was removed under reduced pressure. The residue wasfiltered, and purified by column chromatography to give the titlecompound.

¹HNMR (400 MHz, DMSO-d₆) δppm: 10.11 (s, 1H), 7.64-7.67 (d, 1H),7.21-7.23 (d, 1H), 2.50 (s, 3H).

LC-MS m/z: [M+H]⁺=168.

Step 9: Preparation of(2S)-2-((((6-fluoro-3-methylbenzo[d]isoxazol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 3-methyl-5-hydroxy-6-fluoro-benzo[d]isoxazole preparedfrom Step 8, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (400 MHz, DMSO-d₆) δppm: 11.53 (s, 1H), 7.86 (m, 2H), 7.57 (s,1H), 6.28-6.32 (m, 1H), 5.91 (m, 2H), 5.56-5.602 (m, 1H), 4.82-4.86 (m,1H), 4.31-4.42 (m, 2H), 4.01-4.04 (m, 1H), 3.80-3.85 (m, 2H), 2.52 (s,3H), 1.22-1.29 (m, 6H), 1.12-1.15 (m, 6H).

LC-MS m/z: [M+H]⁺=603.

Example 35:(2S)-2-((((6-fluorobenzo[c][1,2,5]thiadiazol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-fluoro-5-methoxy-2-nitroaniline

3.4 g 2-amino-4,5-difluoronitrobenzene was added to a 100 mLround-bottomed flask, and dissolved by adding 30 mL MeOH. 2.16 g sodiummethoxide was slowly added at room temperature. The mixture was reactedfor another 12 h at room temperature before the reaction was stopped.The reaction solution was filtered, and the filter cake was washed with10 mL cold MeOH, and dried in vacuum to give the title compound, whichwas used directly in the next step.

LC-MS m/z: [M+H]⁺=187.

Step 2: Preparation of 4-fluoro-5-methoxy-1,2-phenylenediamine

2.7 g 4-fluoro-5-methoxy-2-nitroaniline prepared from Step 1 was addedto a 100 mL round-bottomed flask, 30 mL MeOH was added, and 270 mg Pd/Cwas slowly added at room temperature. The mixture was reacted for 3 h at50° C. under hydrogen atmosphere before the reaction was stopped. Thereaction solution was filtered, and the filter cake was washed with coldMeOH (10 mL×2). The filtrate was concentrated to give the titlecompound, which was used directly in the next step.

LC-MS m/z: [M+H]⁺=157.

Step 3: Preparation of 5-fluoro-6-methoxybenzo[c][1,2,5]thiadiazole

1.5 g 4-fluoro-5-methoxy-1,2-phenylenediamine prepared from Step 2 wasadded to a 100 mL round-bottomed flask and dissolved by adding 20 mLpyridine. 2.36 g SOCl₂ was slowly added at 0° C. The mixture was reactedfor 12 h at 50° C. under nitrogen protection before the reaction wasstopped. The reaction solution was poured into 10 g ice, and extractedwith ethyl acetate. The organic layers were collected, washedsuccessively with water, 0.5N cold diluted hydrochloric acid andsaturated brine, dried, concentrated, and purified by columnchromatography to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 7.99-8.02 (d, 1H), 7.65-7.67 (d, 1H),4.02 (s, 3H).

LC-MS m/z: [M+H]⁺=185.

Step 4: Preparation of 5-hydroxy-6-fluorobenzo[c][1,2,5]thiadiazole

500 mg 5-fluoro-6-methoxybenzo[c][1,2,5]thiadiazole prepared from Step 3was added to a reaction flask, and 4 mL 40% HBr aqueous solution wasadded. The mixture was reacted for 12 h at 90° C. under a sealedcondition before the reaction was stopped. The reaction solution wascooled to room temperature and filtered. The filter cake was washed withwater and dried in vacuum to give the title compound.

LC-MS m/z: [M+H]⁺=171.

Step 5: Preparation of(2S)-2-((((6-fluorobenzo[c][1,2,5]thiadiazol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 5-hydroxy-6-fluorobenzo[c][1,2,5]thiadiazole preparedfrom Step 4, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (400 MHz, DMSO-d₆) δppm: 11.54-11.56 (m, 1H), 8.08-8.20 (m, 2H),7.57 (m, 1H), 6.48-6.54 (m, 1H), 5.91-6.10 (m, 2H), 5.52-5.18 (m, 1H),4.78-4.85 (m, 1H), 4.35-4.38 (m, 2H), 3.81-4.07 (m, 3H), 1.16-1.30 (m,6H), 1.11-1.13 (m, 6H).

LC-MS m/z: [M+H]⁺=606.

Example 36:(2S)-2-(((((4′-methoxy-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-4′-methoxy-1,1′-biphenyl, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.49 (s, 1H), 7.61-7.55 (m, 5H), 7.28-7.23 (m,2H), 7.01 (d, 2H), 6.05-6.01 (m, 2H), 5.84-5.82 (m, 1H), 5.57-5.55 (m,1H), 4.88-4.84 (m, 1H), 4.41-4.37 (m, 1H), 4.27-4.25 (m, 1H), 4.05-4.01(m, 1H) 3.84-3.82 (m, 2H), 2.333.79 (s, 3H) 1.28-1.22 (m, 6H), 1.18-1.15(m, 6H).

ESI-MS m/z: [M+H]⁺=636.1.

Example 37: Preparation of(2S)-2-(((((4′-trifluoromethyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-hydroxy-4′-trifluoromethyl-[1,1′-biphenyl]

2 g 4-iodobenzotrifluoride and 1.2 g p-hydroxyphenylboronic acid wereadded to a 100 mL single-neck flask, and 2 g potassium carbonate, 0.26 gPdCl₂(PPh₃)₂, 30 mL 1,4-dioxane and 10 mL water were added. The mixturewas reacted for 4 h at 70° C. under nitrogen protection before thereaction was stopped. The resultant mixture was concentrated, and 10 mLwater was added. The mixture was extracted with ethyl acetate (2×10 mL),dried, filtered, concentrated, and purified by column chromatography togive the title compound.

Step 2: Preparation of(2S)-2-(((((4′-trifluoromethyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-4′-trifluoromethyl-[1,1′-biphenyl]preparedfrom Step 1, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.49 (s, 1H), 7.87 (d, 2H), 7.80 (d, 2H), 7.76(d, 2H), 7.57 (d, 1H), 7.36 (d, 2H), 6.11-6.01 (m, 2H), 5.88-5.83 (m,1H), 5.60-5.55 (m, 1H), 4.89-4.84 (m, 1H), 4.42-4.39 (m, 1H), 4.28-4.26(m, 1H), 4.04-4.02 (m, 1H), 3.85-3.84 (m, 2H), 1.28-1.24 (m, 6H),1.16-1.14 (m, 6H).

ESI-MS m/z: [M+H]⁺=674.

Example 38:(2S)-2-(((4-(thiazol-4-yl)phenyloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-(thiazol-4-yl)phenol

The title compound was prepared according to the method described inStep 1 of Example 37 using p-hydroxyphenylboronic acid, 4-bromothiazole,potassium carbonate and bis(triphenylphosphine)palladium dichloride asstarting materials.

¹HNMR (300 MHz, CDCL3-d₆) δppm: 8.65 (s, 1H), 7.83 (s, 12H), 7.39 (s,1H), 6.90 (d, 2H), 5.00 (S, 1H).

Step 2: Preparation of(2S)-2-(((4-(thiazol-4-yl)phenyloxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-(thiazol-4-yl)phenol prepared from Step 1, phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.48 (s, 1H), 9.18 (s, 1H), 8.12 (s, 1H), 8.00(d, 2H), 7.58 (s, 1H), 7.31 (d, 2H), 6.08-6.04 (m, 2H), 5.82 (d, 1H),5.57 (m, 1H), 4.87-4.85 (m, 1H), 4.38-4.37 (m, 1H), 4.27 (m, 1H), 4.02(m, 1H), 3.84-3.82 (m, 2H), 1.28-1.24 (m, 6H), 1.16-1.14 (m, 6H).

ESI-MS m/z: [M+H]⁺=613.

Example 39:(2S)-2-(((((4′-chloro-2′-fluoro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-hydroxy-4′-chloro-2′-fluoro-[1,1′-biphenyl]

The title compound was prepared according to the method described inStep 1 of Example 37 using 2-fluoro-4-chloroiodobenzene,p-hydroxyphenylboronic acid, potassium carbonate and PdCl₂(PPh₃)₂ asstarting materials.

¹HNMR (300 MHz, CDCL3-d₆) δppm: 7.43-6.83 (m, 7H), 4.94 (s, 1H).

Step 2: Preparation of(2S)-2-(((((4′-chloro-2′-fluoro-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-4′-chloro-2′-fluoro-[1,1′-biphenyl]preparedfrom Step 1, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.48 (s, 1H), 7.56-7.52 (m, 5H), 7.39-7.30 (m,3H), 6.11-6.01 (m, 2H), 5.86-5.82 (m, 1H), 5.56-5.54 (m, 1H), 4.87-4.85(m, 1H), 4.42-4.39 (m, 1H), 4.28-4.26 (m, 1H), 4.04-4.02 (m, 1H)3.85-3.84 (m, 2H), 1.28-1.24 (m, 6H), 1.16-1.14 (m, 6H).

ESI-MS m/z: [M+Na]+=680.

Example 40:(2S)-2-(((((3′-fluoro-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-hydroxy-3′-fluoro-4′-methyl-[1,1′-biphenyl]

The title compound was prepared according to the method described inStep 1 of Example 37 using 3-fluoro-4-methyl-iodobenzene,p-hydroxyphenylboronic acid, potassium carbonate and PdCl₂(PPh₃)₂ asstarting materials.

¹HNMR (300 MHz, CDCL3-d₆) δppm: 7.45-6.87 (m, 7H), 4.77 (s, 1H), 2.31(s, 3H).

Step 2: Preparation of(2S)-2-(((((3′-fluoro-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-3′-fluoro-4′-methyl-[1,1′-biphenyl]preparedfrom Step 1, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.48 (s, 1H), 7.68 (d, 2H), 7.57 (d, 1H),7.44-7.29 (m, 5H) 6.08-6.03 (m, 2H), 5.85-5.82 (m, 1H), 5.56-5.54 (m,1H), 4.88-4.83 (m, 1H), 4.42-4.38 (m, 1H), 4.28-4.26 (m, 1H), 4.04-4.02(m, 1H), 3.84-3.82 (m, 2H), 2.26 (d, 3H), 1.28-1.23 (m, 6H), 1.19-1.14(m, 6H).

ESI-MS m/z: [M+Na]+=660.

Example 41:(2S)-2-((((4′-fluoro-3′-methyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

Step 1: Preparation of 4-hydroxy-4′-fluoro-3′-methyl-[1,1′-biphenyl]

The title compound was prepared according to the method described inStep 1 of Example 37 using 4-fluoro-3-methyl-iodobenzene,p-hydroxyphenylboronic acid, potassium carbonate and PdCl₂(PPh₃)₂ asstarting materials.

¹H NMR (300 MHz, CDCL3-d₆) δ ppm: 7.52-7.42 (m, 2H), 7.34-7.26 (m, 2H),7.06-6.87 (m, 3H), 4.67 (s, 1H), 2.32 (s, 3H).

Step 2: Preparation of(2S)-2-((((4′-fluoro-3′-methyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-4′-fluoro-3′-methyl-[1,1′-biphenyl]preparedfrom Step 1, phosphorus oxychloride, L-alanine isopropyl esterhydrochloride, pentafluorophenol and(2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.50 (s, 1H), 7.65-7.55 (m, 4H), 7.47-7.45 (m,1H), 7.30-7.17 (m, 3H), 6.11-5.99 (m, 2H), 5.85-5.83 (m, 1H), 5.57-5.54(m, 1H), 4.90-4.84 (m, 1H), 4.38-4. 36 (m, 1H), 4.27-4.25 (m, 1H),4.02-3.99 (m, 1H), 3.87-3.80 (m, 2H), 2.30 (s, 3H), 1.29-1.21 (m, 6H),1.16-1.14 (m, 6H).

ESI-MS m/z: [M+Na]⁺=660.

Example 42:(2S)-2-((((4′-methyl-[1,1′-biphenyl]-4-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the method described inExample 1 using 4-hydroxy-4′-methyl-[1,1′-biphenyl], phosphorusoxychloride, L-alanine isopropyl ester hydrochloride, pentafluorophenoland (2′R)-2′-deoxy-2′-fluoro-2′-methyluridine as starting materials.

¹HNMR (500 MHz, DMSO) δ: 11.49 (s, 1H), 7.58 (d, 1H), 7.53-7.51 (m, 4H),7.29-7.25 (m, 4H), 6.10-6.02 (m, 2H), 5.88-5.82 (m, 1H), 5.60-5.55 (m,1H), 4.88-4.84 (m, 1H), 4.41-4.38 (m, 1H), 4.27 (m, 1H), 4.04-4.02 (m,1H) 3.84-3.82 (m, 2H), 2.33 (s, 3H), 1.28-1.23 (m, 6H), 1.18-1.14 (m,6H).

ESI-MS m/z: [M+H]⁺=620.

Example 43:(2S)-2-(((R)-((4-fluoro-1,2-dimethyl-1H-indol-5-yl)oxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)amino)propanoicAcid Isopropyl Ester

The title compound was prepared according to the similar method asdescribed in Example 33.

¹HNMR (400 MHz, DMSO-d₆) δ: 11.49 (s, 1H), 7.55-7.57 (d, 1H). 7.07-7.19(m, 2H), 6.27 (s, 1H), 5.91-6.11 (m, 3H), 5.14-5.56 (d, 1H), 4.82-4.88(t, 1H), 4.41-4.45 (m, 1H), 4.24-4.28 (m, 1H), 4.04-4.06 (m, 1H),3.74-3.82 (m, 2H), 3.647 (s, 3H), 2.39 (s, 3H), 1.20-1.25 (m, 6H),1.14-1.16 (m, 6H).

LC-MS m/z: [M+H]⁺=615.

Pharmacological and Pharmacokinetic Activity Experimental Example 1:Antiviral Activity Assay of the Compounds of the Present Invention inHCV Replicon System

1. Experimental Materials

1.1 Reagents:

TABLE 1 List of reagents Name of reagents Supplier DMEM mediumInvitrogen Fetal bovine serum (FBS) Gibco L-Glutamine Invitrogenpenicillin-streptomycin solution Invitrogen DPBS/Modified HycloneTrypsin/EDTA Invitrogen Dimethyl sulfoxide (DMSO) Sigma Bright-Glo ™Promega CellTiter-Fluor ™ Reagent Promega1.2 Huh7 1b Cell Lines:

The cell line Huh7 1b, which was provided by WuXi AppTec Co., Ltd. inShanghai, contained an HCV 1b replicon with a stable Luciferase (Luc)reporter. It was constructed by cloning HCV nonstructural protein gene,neo (G418 resistance) and luciferase reporter gene into the pBR vectorthrough gene recombination technology. Then the vector carrying the HCVreplicon was transfected into Huh7 cells, and the transfected Huh7 cellswere screened by G418 resistance. The HCV replicon was stablyreplicated, and the related protein and luciferase were stably expressedin Huh7 cells. The cells model was used for in-vitro screen of anti-HCVcompounds. The anti-HCV activity of the compounds was measured bydetecting the level of luciferase expression. See Lohmann V, et al.1999. Replication of subgenomic hepatitis C virus RNAs in a hepatomacell line. Science. 285(5424):110-113.

1.3 Positive Control:

The control drug used in this experimental example was the compound ofExample 25 in WO 2008/121634 (PCT/US2008/058183), i.e.(S)-2-{[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]-phenoxy-phosphorylamino}-propanoicacid isopropyl ester. This compound was prepared according to the methoddescribed in J. Org. chem, 2011, 76, 8311-8319, and identified by H-NMRand mass spectrometry.

2. Experimental Procedures:

-   -   2.1 compound preparation: adding each of the compounds listed in        Table 2 to 96-well plates using a POD™ 810 Automation Platform        (LabCyte Corporation, USA), with 10 μM of each of the compounds        as a starting concentration and in duplicate; serially diluting        each of the compounds in 3-fold fashion and formulating 10        concentrations, with the final concentration of DMSO being 0.5%;    -   2.2 cells preparation: seeding the Huh71b cells into 96-well        plates respectively with a volume of 125 μl and 8×10³        cells/well, and incubating in a 37° C., 5% CO₂ incubator for 72        h;    -   2.3 detection of cell activity: adding 30 μl per cell of the        CellTiter-Fluor™ Reagent, incubating in a 37° C., 5% CO₂        incubator for 1 h, measuring the fluorescence signal values with        a fluorometer, and using the obtained data for calculation of        cytotoxicities of the compounds;    -   2.4 Bright-Glo detection: adding 100 μl per well of the        Bright-Glo™ Luciferase Substrate, using a chemiluminescence        detection system EnVision™ (PerkinElmer, USA) to detect the        fluorescence signal values within 5 minutes, and using the        obtained data for calculation of potencies of the compounds.    -   2.5 data processing: converting the obtained data to a        percentage of cell viability (Viability %) using the following        formula:

${{Viability}\mspace{14mu}\%} = {\frac{CPD}{ZPE} \times 100}$

-   -   -   CPD: fluorescence signal value of the well added with the            compound        -   ZPE (Zero percent effect): fluorescence signal value of the            control well with zero percent effect.        -   Processing the raw data to calculate a percentage of            inhibition (Inhibition %) using the following formula:

${{Inhibition}\mspace{14mu}\%} = {\frac{{CPD} - {HPE}}{{ZPE} - {HPE}} \times 100}$

-   -   -   CPD: fluorescence signal value of the well added with the            compound;        -   HPE (Hundred percent effect): fluorescence signal value of            the control well with hundred percent effect;        -   ZPE (Zero percent effect): fluorescence signal value of the            control well with zero percent effect.

The inhibition percentage was processed by the GraphPad Prism softwareto obtain the corresponding curves and EC₅₀ values. The data was listedin Table 2.

TABLE 2 Example Number Structure EC₅₀ (μM) Positive control

0.1168 Example 1

0.056 Example 2

0.1051 Example 3

0.1232 Example 4

undetected Example 5

0.061 Example 6

undetected Example 7

undetected Example 8

0.411 Example 9

undetected Example 10

undetected Example 11

0.189 Example 12

0.637 Example 13

undetected Example 14

0.2231 Example 15

>3.33 Example 16

3.871 Example 17

0.2577 Example 18

0.3141 Example 19

0.303 Example 20

3.871 Example 21

0.2577 Example 22

0.3141 Example 23

4.488 Example 24

undetected Example 25

undetected Example 27

0.098 Example 28

0.123 Example 31

0.102 Example 32

0.068 Example 33

0.033 Example 36

0.073 Example 38

0.100 Example 39

0.061 Example 40

0.056 Example 41

0.050 Example 42

0.126 Example 43

0.069

The above results have shown that the compounds of the present inventionhave the ability to inhibit HCV virus very efficiently, and compared tothe positive control, have a superior or equal activity based on EC₅₀values.

Experimental Example 2: Antiviral Activity Assay of the Compounds of thePresent Invention in the Cell-Culture-Derived Infectious HCV (HCVcc)System

1. Experimental Materials

1.1 Compounds

The compound according to the above example 1 was used in thisexperiment. The compound was formulated into a 10 mM mother liquid withDMSO, diluted to 500 nM by DMEM complete culture medium containing 0.5%of DMSO, followed by 4-fold dilution successively, and sixconcentrations were obtained. The positive control was diluted to 10 μMby DMEM complete culture medium containing 0.5% of DMSO, followed by4-fold successively, and six concentrations were obtained.

1.2 Cells

Huh 7.5.1 cells, provided by Shanghai Institute of Materia Medica,Chinese Academy of Sciences.

1.3 Viruses

J399EM (HCV genotype 2a) virus, i.e. a full length HCV mutant straintransfected with EGFP (enhanced green fluorescent protein) was used,which had the same infection capacity as the wild type JFH-1. TheNS5A-EGFP fusion protein fluorescence was observed directly in infectedcells by inserting EGFP coding sequence in the NS5A region. J399EM wasprovided by Shanghai Institute of Materia Medica, Chinese Academy ofSciences.

1.4 Reagents

DMEM medium, purchased from Invitrogen Corporation, USA;

Fetal bovine serum (FBS), purchased from Sigma Corporation, USA;

L(+)-Glutamine, purchased from Invitrogen Corporation, USA;

Penicillin-streptomycin (Pen-Strep), purchased from InvitrogenCorporation, USA;

Phosphate buffered saline (PBS), purchased from Hyclone Corporation,USA;

Trypsin, purchased from Invitrogen Corporation, USA;

Dimethyl sulfoxide (DMSO), purchased from Sigma Corporation, USA;

Lysis buffer, purchased from Promega Corporation, USA;

MTT, purchased from Sigma Corporation, USA.

1.4 Instruments

EnVision® MultilabelPlate readers, purchased from Perkin-ElmerCorporation, USA.

2 Experimental Procedures

-   1) Huh 7.5.1 cells preparation: collecting the Huh7.5.1 cells in    logarithmic phase, resuspending in DMEM complete culture medium,    seeding into 96-well plates (7×10³ cells/well), and incubating in a    37° C., 5% CO₂ incubator for 24 h;-   2) virus infection: resuspending J399EM viruses in DMEM complete    culture medium, adding the virus supernatant (MOI≈0.1) to the above    96-well plates, and washing with PBS after 8 hours of infection;-   3) drug treatment: adding various concentrations of the compounds to    the Huh7.5.1 cells infected by J399EM viruses, with each    concentration in duplicate; setting the control group with zero    percent effect (ZPE) and the control group with hundred percent    effect (HPE); replacing the compound with the DMEM complete culture    medium containing 0.5% DMSO in ZPE group, and using the cells not    infected with viruses in HPE group.-   4) cells incubation: incubating the 96-well plates in a 37° C., 5%    CO₂ incubator for 72 hr;-   5) anti-HCV activity detection: reading the relative fluorescence    units (RFU) of each well by EnVision® Multi label Plate readers    after incubation, and calculating the anti-HCV activity of the    compounds using the obtained data according to the following    formula:    Inhibition %=(RFU_(ZPE)−RFU_(CPD))/(RFU_(ZPE)−RFU_(HPE))×100    -   wherein RFU_(ZPE) represents the relative fluorescence units of        the control group with zero percent effect and RFU_(CPD)        represents the relative fluorescence units of the corresponding        compound group, and RFU_(HPE) represents the relative        fluorescence units of the control group with hundred percent        effect.-   6) Cell viability detection: adding MTT solution to each well,    incubating in a 37° C., 5% CO₂ incubator for 4 hr, followed by    addition of MTT dissolving solution, reading optical density (OD) of    each well at 570 nm by the Multi label Plate readers after 6 hr,    calculating the cytotoxicity of the compounds using the obtained    data according to the following formula:    Viability %=OD_(CPD)/OD_(ZPE)×100    -   wherein OD_(ZPE) represents the optical density of the control        group with zero percent effect, and OD_(CPD) represents the        optical density of the corresponding compound;-   7) Data processing: processing the Inhibition %, Viability % by    GraphPad Prism Software respectively, and obtaining the half maximal    effective concentration (EC₅₀) values and half maximal cytotoxic    concentration (CC₅₀) values of the compounds against the HVCcc GT2a    viruses. The results were listed in Table 3.

TABLE 3 Example EC₅₀ CC₅₀ Number Structure (μM) (μM) Positive control

0.173 >10 Example 1

0.039 >10

It was seen from Table 3 that the compound of Example 1 of the presentinvention had excellent antiviral activity and less cell toxicitycompared with the positive control in the HCVcc system.

In addition, the experiments of the present invention also showed thatusing in vitro HCVcc system, the compounds prepared by the presentinvention, such as the compounds prepared in Examples 2, 5, 12, 16, 18,28 and 38, had a low half maximal effective concentration (EC₅₀) and ahigh half maximal cytotoxic concentration (CC₅₀) against the HCVcc GT2aviruses, which demonstrated good inhibitory activity and smallcytotoxicity.

Experimental Example 3: Pharmacokinetic Studies of the Compounds of thePresent Invention in SD Rats

1. Experimental Materials

1.1 Compounds

In this experiment, the compound of Example 1 was used, and the positivecontrol drug was the same as above. Each of the compounds was added to0.5% sodium carboxymethyl cellulose (CMC), and the mixture was vortexedto prepare a 10 mg/mL suspension for intragastric administration.

The standard substance GS-461203 was the metabolite of the testedcompounds, and the chemical name was(2′R)-2′-deoxy-2′-fluoro-2′-methyluridinetriphosphate, which waspurchased from TriLink BioTechnologies Corporation, USA.

1.2 Animals

Male SD rats, 6-8 weeks old, weighing 237.0-268.4 g, provided byShanghai Super-B&K laboratory animal Co. Limited.

1.3 Reagents

methanol (chromatographic pure), purchased from Spectrum Corporation,USA;

acetonitrile (chromatographic pure): purchased from SpectrumCorporation, USA.

1.4 Instruments

API 5500 LC-MS, purchased from AB Corporation, USA.

2. Experimental method

-   1) administration: dividing SD rats into 4 groups, with 18 rats in    each group, intragastrically administering with the compound in an    amount of 50 mg/kg. Fasting the rats for 10-15 hours prior to    intragastric administration, and refeeding four hours after    administration;-   2) sampling: at 0.5 h, 1 h, 2 h, 4 h, 6 h and 12 h after    administration, sacrificing the rats (n=3 at each time point for    each group), collecting about 1 g of each rat liver respectively,    adding 3 volumes of pre-cooled methanol, homogenizing for 30 s and    storing the prepared liver homogenate in −80° C. before analysis.-   3) standard curve preparation: taking appropriate amount of    GS-461203 stock solution, serially diluting with methanol to prepare    standard solutions with concentrations of 30, 27, 10, 5, 2, 1, 0.5,    0.2 and 0.1 μg/mL, parallelly adding 10 μL of each standard solution    to 90 μL blank rat liver homogenate to prepare sample standard    curves of concentrations of 3000, 2700, 1000, 500, 200, 100, 50, 20    and 10 ng/mL.-   4) sample processing: adding 30 μl liver homogenate sample or    standard curve sample to 150 μl solution of acetonitrile containing    the internal standard (100 ng/mL), vortexing for 2 min, centrifuging    for 10 min (6000 revolutions/min), and transferring the supernatant    to injection vials;-   5) sample data analysis: taking 5 μL supernatant, injecting, and    then using UPLC-MS/MS to detect the concentration of GS-461203 in    the samples; according to the obtained drug concentration-time data    of the liver homogenates, using the non-compartmental model of    pharmacokinetic calculation software WinNonlin 6.2.1 to calculate    pharmacokinetic parameters of metabolite GS-461203 of each of the    tested compounds. The results were listed in Table 4.

TABLE 4 GS-461203 C_(max) T_(max) AUC_(last) (ng/g liver) (hr) (ng/g*hr)Positive control 753 2.00 5627 Example 1 818 1.00 7106

It was seen from the data in Table 4 that the compound of Example 1according to the present invention was better than that of the positivecontrol in the peak concentration in liver tissue and the area under theplasma drug concentration-time curve of the metabolite, and had greaterin vivo exposure level.

The above results show that the compounds of the present invention havethe ability to efficiently inhibit HCV virus, and compared to thepositive control, have equal or superior effects in EC₅₀, less toxicityto the host cells, higher CC₅₀, and good security. The compounds of thepresent invention have good prospects in the treatment of HCV infection.

Although the present invention has been described in detail above, itshould be understood by those skilled in the art that variousmodifications and alterations can be made without departing from thespirit and scope of the present invention. The scope of the presentinvention is not limited to the foregoing detailed description and isdefined by the claims.

What is claimed is:
 1. A nucleoside phosphoramidate compound of thefollowing general formula I,

or a salt, or crystal thereof, wherein (1) R₁ is selected from C₁₋₆alkyl; (2) R₂ is selected from halogen; (3) R₃ is selected from OH, Hand C₁₋₄ alkoxy; (4) R₄ is selected from H, C₁₋₆ alkyl and halogenatedC₁₋₆ alkyl; (5) R₅ is selected from C₁₋₆ alkyl and halogenated C₁₋₆alkyl; (6) R₆ is selected from the following moieties: a) phenyl-Y—,wherein Y is absent or selected from C₁₋₆ alkylenyl, C₂₋₆ alkenylenyl,C₂₋₆ alkynylenyl, C₂₋₆ alkenylenyl-(CO)—, C₂₋₆ alkynylenyl-(CO)—, O, S,NH— and —N(C₁₋₆ alkyl), and wherein the phenyl is optionally substitutedby one or more groups selected from C₁₋₆ alkyl, halogen, nitro, C₁₋₆alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino, halogenatedC₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆alkyl NHCO, or the phenyl and a group selected from phenyl, oxazolyl,pyrazinyl and pyrrolyl taken together form a naphthyl, benzoxazolyl,benzo[b]pyrazinyl or benzo[b]pyrrolyl; b) heterocyclyl-Y—, which isselected from the group consisting of 1H-imidazolyl-Y—,1,2,4-triazolyl-Y—, 1,2,3-triazolyl-Y—, thiazolyl-Y—,1,2,3-thiadiazolyl-Y—, 1,2,4-thiadiazolyl-Y—, 1,3,4-thiadiazolyl-Y—,oxazolyl-Y—, 1,2,4-oxadiazolyl-Y—, 1,2,3-oxadiazolyl-Y—,1,3,4-oxadiazolyl-Y—, pyrimidinyl-Y—, pyrazinyl-Y—, pyridazinyl-Y—,quinoxalinyl-Y—, 4H-chromen-4-one-Y—, pyridyl-Y—, thienyl-Y—, andthieno[3,2-b]thienyl-Y—, wherein Y is absent or selected from C₁₋₆alkylenyl, C₂₋₆ alkenylenyl, C₂₋₆ alkynylenyl, C₂₋₆ alkenylenyl-(CO)—,C₂₋₆ alkynylenyl-(CO)—, O, S, —NH— and —N(C₁₋₆ alkyl) and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₆ alkyl, halogen, nitro, C₁₋₆ alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₁₋₆ acylamino, halogenated C₁₋₆ alkyl, halogenated C₁₋₆alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆ alkyl NHCO; c) C₁₋₆alkyl-O—C(O)—C₂₋₆ alkenylenyl- and C₁₋₆ alkyl-O—C(O)—C₂₋₆alkenylenyl-C(O)—, wherein the C₁₋₆ alkyl is optionally substituted byone or more groups selected from C₁₋₆ alkyl, halogen, nitro, C₁₋₆alkoxy, cyano, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ acylamino, halogenatedC₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, amino, N(C₁₋₆ alkyl)₂ and C₁₋₆alkyl NHCO; and (7) R₇ is selected from H, halogen, C₁₋₆ alkyl,halogenated C₁₋₆ alkyl, C₁₋₆ alkoxy, halogenated C₁₋₆ alkoxy, NO₂, CN,C₁₋₆ alkyl-NH—CO—, hydroxy, mono-C₁₋₆ alkylamino, di-C₁₋₆ alkylamino,C₁₋₆ alkyl-S—, C₂₋₆ alkenyl-S—, C₂₋₆ alkynyl-S—, C₁₋₆ alkyl-SO—, C₂₋₆alkenyl-SO—, C₂₋₆ alkynyl-SO—, C₁₋₆ alkyl-SO₂—, C₂₋₆ alkenyl-SO₂—, C₂₋₆alkynyl-SO₂—, C₁₋₆ alkyl-OSO₂—, C₂₋₆ alkenyl-OSO₂—, and C₂₋₆alkenyl-OSO₂—; or (8) R₆ and R₇ together with the benzene ring to whichthey are attached form a benzo-annelated five-membered ring orbenzo-annelated six-membered ring selected from the group consisting of1,2,3,4-tetrahydronaphthalene, 2,3-dihydro-1H-indene, indole,benzofuran, quinoxaline, 4H-chromen-4-one, benzo[d]isoxazole,benzo[d]oxazole, benzo[c][1,2,5]thiadiazole, benzo[b]thiophene, andbenzodihydropyran-4-one, wherein the benzo-annelated five-membered ringor benzo-annelated six-membered ring is optionally substituted by one ormore groups selected from halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogenatedC₁₋₆ alkyl, halogenated C₁₋₆ alkoxy, phenyl, cyano, C₁₋₆ alkyl-OC(O)—and C₁₋₆ alkyl-OC(O)—CH₂—.
 2. The compound according to claim 1, or thesalt or crystal thereof, wherein R₁ is selected from C₁₋₃ alkyl, R₂ isF, R₃ is selected from OH, H and methoxy, R₄ is selected from H and C₁₋₆alkyl, and R₅ is selected from C₁₋₆ alkyl.
 3. The compound according toclaim 2, or the salt or crystal thereof, wherein R₆ is selected from a)phenyl-Y—, wherein Y is absent or selected from C₁₋₄ alkylenyl, C₂₋₄alkenylenyl, C₂₋₄ alkynylenyl, C₂₋₄ alkenylenyl-(CO)—, C₂₋₄alkynylenyl-(CO)—, O, S, —NH— and —N(C₁₋₄ alkyl), and wherein the phenylis optionally substituted by one or more groups selected from C₁₋₄alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl,C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino,N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO, or the phenyl and a group selectedfrom phenyl, oxazolyl, pyrazinyl and pyrrolyl taken together form anaphthyl, benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl; b)heterocyclyl-Y—, which is selected from the group consisting of1H-imidazolyl-Y—, 1,2,4-triazolyl-Y—, 1,2,3-triazolyl-Y—, thiazolyl-Y—,1,2,3-thiadiazolyl-Y—, 1,2,4-thiadiazolyl-Y—, 1,3,4-thiadiazolyl-Y—,oxazolyl-Y—, 1,2,4-oxadiazolyl-Y—, 1,2,3-oxadiazolyl-Y—,1,3,4-oxadiazolyl-Y—, pyrimidinyl-Y—, pyrazinyl-Y—, pyridazinyl-Y—,quinoxalinyl-Y—, 4H-chromen-4-one-Y—, pyridyl-Y—, thienyl-Y—, andthieno[3,2-b]thienyl-Y—, wherein Y is absent or selected from C₁₋₄alkylenyl, C₂₋₄ alkenylenyl, C₂₋₄ alkynylenyl, C₂₋₄ alkenylenyl-(CO)—,C₂₋₄ alkynylenyl-(CO)—, O, S, —NH— and —N(C₁₋₄ alkyl), and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; and c) C₁₋₄alkyl-O—C(O)—C₂₋₄ alkenylenyl- and C₁₋₄ alkyl-O—C(O)—C₂₋₄alkenylenyl-C(O)—, wherein the C₁₋₄ alkyl is optionally substituted byone or more groups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenatedC₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄alkyl NHCO.
 4. The compound according to claim 3, or the salt or crystalthereof, wherein R₆ is selected from a) phenyl, phenyl-C₁₋₃ alkylenyl-,phenyl-C₂₋₃ alkenylenyl-, phenyl-C₂₋₃ alkynylenyl-, phenyl-O—,phenyl-S—, phenyl-NH—, phenyl-N(C₁₋₃ alkyl)-, phenyl-ethenylenyl-(CO)—and naphthyl-ethenylenyl-(CO)—, wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₄ alkyl, halogen,nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino,halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂and C₁₋₄ alkyl NHCO, or the phenyl and a group selected from phenyl,oxazolyl, pyrazinyl and pyrrolyl taken together form a naphthyl,benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl; b)1H-imidazolyl-Y—, 1,2,4-triazolyl-Y—, 1,2,3-triazolyl-Y—, thiazolyl-Y—,1,2,3-thiadiazolyl-Y—, 1,2,4-thiadiazolyl-Y—, 1,3,4-thiadiazolyl-Y—,oxazolyl-Y—, 1,2,4-oxadiazolyl-Y—, 1,2,3-oxadiazolyl-Y—,1,3,4-oxadiazolyl-Y—, pyrimidinyl-Y—, pyrazinyl-Y—, pyridazinyl-Y—,quinoxalinyl-Y—, 4H-chromen-4-one-Y—, pyridyl-Y—, thienyl-Y—,thieno[3,2-b]thienyl-Y—, wherein Y is absent or selected frommethylenyl, ethylenyl, ethenylenyl, ethynylenyl, ethenylenyl-(CO)—,ethynylenyl-(CO)—, O, S, —NH— and —NCH₂—, wherein each of theheterocyclyl groups is optionally substituted by one or more groupsselected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; andc) methyl-OC(O)—═—, ethyl-OC(O)—═—, propyl-OC(O)—═—, isopropyl-OC(O)—═—,butyl-OC(O)—═—, isobutyl-OC(O)—═—, and t-butyl-OC(O)—═—.
 5. The compoundaccording to claim 2, or the salt or crystal thereof, wherein R₆ group“phenyl-Y—” or “heterocyclyl-Y—” and the oxygen group both of which areattached to the benzene ring are at the para- or meta-position to eachother.
 6. The compound according to claim 2, or the salt or crystalthereof, wherein R₁ is CH₃, R₂ is F, R₃ is OH, R₄ is CH₃, and R₅ isisopropyl.
 7. The compound according to claim 1, or the salt or crystalthereof, wherein R₆ is selected from a) phenyl-Y—, wherein Y is absentor selected from C₁₋₄ alkylenyl, C₂₋₄ alkenylenyl, C₂₋₄ alkynylenyl C₂₋₄alkylenyl-(CO)—, C₂₋₄ alkynylenyl-(CO)—, O, S, —NH— and —N(C₁₋₄ alkyl),and wherein the phenyl is optionally substituted by one or more groupsselected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO, orthe phenyl and a group selected from phenyl, oxazolyl, pyrazinyl andpyrrolyl taken together form a naphthyl, benzoxazolyl, benzo[b]pyrazinylor benzo[b]pyrrolyl; b) heterocyclyl-Y—, which is selected from thegroup consisting of 1H-imidazolyl-Y—, 1,2,4-triazolyl-Y—,1,2,3-triazolyl-Y—, thiazolyl-Y—, 1,2,3-thiadiazolyl-Y—,1,2,4-thiadiazolyl-Y—, 1,3,4-thiadiazol-Y—, oxazolyl-Y—,1,2,4-oxadiazolyl-Y—, 1,2,3-oxadiazolyl-Y—, 1,3,4-oxadiazolyl-Y—,pyrimidinyl-Y—, pyrazinyl-Y—, pyridazinyl-Y—, quinoxalinyl-Y—,4H-chromen-4-one-Y—, pyridyl-Y—, thienyl-Y—, andthieno[3,2-b]thienyl-Y—, wherein Y is absent or selected from C₁₋₄alkylenyl, C₂₋₄ alkenylenyl, C₂₋₄ alkynylenyl, C₂₋₄ alkenylenyl-(CO)—,C₂₋₄ alkynylenyl-(CO)—, O, S, —NH— and —N(C₁₋₄ alkyl), and wherein theheterocyclyl is optionally substituted by one or more groups selectedfrom C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl, halogenated C₁₋₄alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; and c) C₁₋₄alkyl-O—C(O)—C₂₋₄ alkenylenyl- and C₁₋₄ alkyl-O—C(O)—C₂₋₄alkenylenyl-C(O)—, wherein the C₁₋₄ alkyl is optionally substituted byone or more groups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenatedC₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄alkyl NHCO.
 8. The compound according to claim 7, or the salt or crystalthereof, wherein R₆ is selected from a) phenyl, phenyl-C₁₋₃ alkylenyl-,phenyl-C₂₋₃ alkenylenyl-, phenyl-C₂₋₃ alkynylenyl-, phenyl-O—,phenyl-S—, phenyl-NH—, phenyl-N(C₁₋₃ alkyl)-, phenyl-ethenylenyl-(CO)—and naphthyl-ethenylenyl-(CO)—, wherein the phenyl is optionallysubstituted by one or more groups selected from C₁₋₄ alkyl, halogen,nitro, C₁₋₄ alkoxy, cyano, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino,halogenated C₁₋₄ alkyl, halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂and C₁₋₄ alkyl NHCO, or the phenyl and a group selected from phenyl,oxazolyl, pyrazinyl and pyrrolyl taken together form a naphthyl,benzoxazolyl, benzo[b]pyrazinyl or benzo[b]pyrrolyl; b)1H-imidazolyl-Y—, 1,2,4-triazolyl-Y—, 1,2,3-triazolyl-Y—, thiazolyl-Y—,1,2,3-thiadiazolyl-Y—, 1,2,4-thiadiazolyl-Y—, 1,3,4-thiadiazolyl-Y—,oxazolyl-Y—, 1,2,4-oxadiazolyl-Y—, 1,2,3-oxadiazolyl-Y—,1,3,4-oxadiazolyl-Y—, pyrimidinyl-Y—, pyrazinyl-Y—, pyridazinyl-Y—,quinoxalinyl-Y—, 4H-chromen-4-one-Y—, pyridyl-Y—, thienyl-Y—,thieno[3,2-b]thienyl-Y—, wherein Y is absent or selected frommethylenyl, ethylenyl, ethenylenyl, ethynylenyl, ethenylenyl-(CO)—,ethynylenyl-(CO)—, O, S, —NH— and —NCH₂—, wherein each of theheterocyclyl groups is optionally substituted by one or more groupsselected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; andc) methyl-OC(O)—═—, ethyl-OC(O)—═—, propyl-OC(O)—═—, isopropyl-OC(O)—═—,butyl-OC(O)—═—, isobutyl-OC(O)—═— and t-butyl-OC(O)—═—.
 9. The compoundaccording to claim 8, or the salt or crystal thereof, wherein R₆ isselected from a) phenyl, phenyl-(CH₂)—, phenyl-=-, phenyl-=-C(O)— andphenyl-=-, wherein the phenyl is optionally substituted by one or moregroups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO, orthe phenyl and a group selected from oxazolyl, pyrazinyl and pyrrolyltaken together form a benzoxazolyl, benzo[b]pyrazinyl orbenzo[b]pyrrolyl; b) 1H-imidazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl,thiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,oxazolyl, 1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl,pyrimidinyl, pyrazinyl, pyridazinyl, quinoxalinyl, 4H-chromen-4-one,pyridyl, thieno[3,2-b]thienyl, 1H-imidazolyl-(CH₂)—,1,2,4-triazolyl-(CH₂)—, 1,2,3-triazolyl-(CH₂)—, thiazolyl-(CH₂)—,1,2,3-thiadiazolyl-(CH₂)—, 1,2,4-thiadiazolyl-(CH₂)—,1,3,4-thiadiazolyl-(CH₂)—, oxazolyl-(CH₂)—, 1,2,4-oxadiazolyl-(CH₂)—,1,2,3-oxadiazolyl-(CH₂), 1,3,4-oxadiazolyl-(CH₂)—, pyrimidinyl-(CH₂)—,pyrazinyl-(CH₂)—, pyridazinyl-(CH₂)—, quinoxalinyl-(CH₂)—,4H-chromen-4-one-(CH₂)—, pyridyl-(CH₂)—, thieno[3,2-b]thienyl-(CH₂)—,1H-imidazolyl-=-, 1,2,4-triazolyl-=-, 1,2,3-triazolyl-=-, thiazolyl-=-,1,2,3-thiadiazolyl-=-, 1,2,4-thiadiazolyl-=-, 1,3,4-thiadiazolyl-=-,oxazolyl-=-, 1,2,4-oxadiazolyl-=-, 1,2,3-oxadiazolyl-=-,1,3,4-oxadiazoly-=-, pyrimidinyl-=-, pyridazinyl-=-, pyridazinyl-=-,quinoxalinyl-=-, 4H-chromen-4-one-=-, pyridyl-=-,thieno[3,2-b]thienyl-=-, 1H-imidazolyl-≡-, 1,2,4-triazolyl-≡-,1,2,3-triazolyl-≡-, thiazolyl-≡-, 1,2,3-thiadiazolyl-≡-,1,2,4-thiadiazolyl-≡-, 1,3,4-thiadiazolyl-≡-, oxazolyl-≡-,1,2,4-oxadiazolyl-≡-, 1,2,3-oxadiazolyl-≡-, 1,3,4-oxadiazolyl-≡-,pyrimidinyl-≡-, pyrazinyl-≡-, pyridazinyl-≡-, quinoxalinyl-≡-,4H-chromen-4-one-≡-, pyridyl-≡-, or thieno[3,2-b]thienyl-≡-, whereineach of the heterocyclyl groups is optionally substituted by one or moregroups selected from C₁₋₄ alkyl, halogen, nitro, C₁₋₄ alkoxy, cyano,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ acylamino, halogenated C₁₋₄ alkyl,halogenated C₁₋₄ alkoxy, amino, N(C₁₋₄ alkyl)₂ and C₁₋₄ alkyl NHCO; andc) methyl-OC(O)—═—.
 10. The compound according to claim 7, or the saltor crystal thereof, wherein R₁ is CH₃, R₂ is F, R₃ is OH, R₄ is CH₃, andR₅ is isopropyl.
 11. The compound according to claim 1, or the salt orcrystal thereof, wherein R₆ group “phenyl-Y—” or “heterocyclyl-Y—” andthe phenolic oxygen group both of which are attached to the benzene ringare at the para- or meta-position to each other.
 12. The compoundaccording to claim 1, or the salt or crystal thereof, wherein R₁ is CH₃,R₂ is F, R₃ is OH, R₄ is CH₃, and R₅ is isopropyl.
 13. The compoundaccording to claim 12, or the salt or crystal thereof, wherein R₆ isphenyl or benzyl.
 14. The compound according to claim 1, or the salt orcrystal thereof, wherein the compound is selected from


15. A pharmaceutical composition comprising the compound according toclaim 14, or the salt or crystal thereof, and a pharmaceuticallyacceptable carrier.
 16. A pharmaceutical composition, comprising thecompound according to claim 1, or the salt or crystal thereof and apharmaceutically acceptable carrier.
 17. A method for treatment ofhepatitis C viral infection, comprising administering a therapeuticallyeffective amount of the pharmaceutical composition according to claim 16to a subject in need thereof.
 18. A method for treatment of hepatitis Cviral infection comprising administering a therapeutically effectiveamount of the compound according to claim 1, or the salt or crystalthereof to a subject in need thereof.